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Preface

This laboratory manual describes the determination of water quality indicators that are measured
rather frequently in the laboratory of UNESCO-IHE.
Most of the procedures are based on the APHA / AWWA / WEF Standard Methods for the
Examination of Water & Wastewater.
The methods, mentioned in this manual, are not necessarily the most suitable for your samples. The
choice of the method will depend on the preservation agents used, the sample volume, the matrix of
the sample and/or the concentration of the analyte.
It is advised to discuss all the technical possibilities with the laboratory staff before you start the
research.

Instrumental methods like GLC (gas-liquid chromatography), IC (ion chromatography), ICP (induced
coupled plasma), AAS (atomic absorption spectrometry), TOC ( total organic carbon), UV
spectrometry, ISE (ion-specific electrodes) and other methods are not discussed here.
A special training, given by the staff of the laboratory, is required before these techniques can be
applied by you.

As a part of our Quality Control programme we will ask you, from time to time, to analyse an
“unknown” sample. The result produced by you will be an indication of the reliability of the data you
are producing during your laboratory activities.

According to our safety regulations you must be informed first about the UNESCO-IHE safety
procedures before you are allowed to start any laboratory activity.
A member of the lab staff will give an explanation of the written procedures which will be handed over
to you. Some of the general safety procedures can be found in this manual.

Fred Kruis
Head of UNESCO-IHE Laboratory

October 2007
SAFETY IN THE LABORATORY

1. INTRODUCTION

According to the statutory regulations (laws concerning public safety) the Director of
UNESCO-IHE is responsible for the safety of everyone working in the buildings and on the
property of UNESCO-IHE.

2. ACCESS TO THE LABORATORIES

The laboratories are open from 08.00-19.45 hrs.(office hours), except on Saturdays,
Sundays and public holidays. On Saturdays it will be open from 08.30-12.30 hrs.
In very special cases it is possible to have access to the laboratory outside office hours, however,
no porter and/or first-aid officer will be present in the building.
In such a case, special safety regulations will be explained to you. Discuss this possibility in time
with the head of the laboratory
The labstaff is present from Monday till Friday and their working hours are
from 08.30-17.00 hrs.

3. SAFETY RULES

3.1 Every one should know:


- the general safety rules for working in an laboratory environment
- the escape routes in case of emergency.
- the regulations for the discharge of waste.

3.2 Eating, drinking and smoking are prohibited in all laboratories.

3.3 Wear always a laboratory coat during chemical or microbiological work.

3.4 Wear safety glasses and gloves when working with aggressive or toxic chemicals.

3.5 Use rubber teats during pipetting of harmful liquids.

3.6. Read the characteristics, the risks and preventive measures of any unknown compound in
the "(Chemical) Safety Sheets". Everyone should know the exact functioning and possible
risks of chemicals and instruments before it is used.

3.7 Use the fume cupboards when toxic, noxious and dangerous solutions are used.
Storage of other materials in fume cupboards is forbidden!

3.8 Hazardous work should not be undertaken outside the regular working hours, there should always
be a second person within shouting distance and in principle in the same room

3.9 Place a sheet with the text "Please, let this apparatus switched ON!" where equipment is left in
operation outside the regular opening hours.

3.10 Good hygiene should be practised with biological material. Materials of human origin, cultures of
micro-organisms etc., should be considered contaminated (meaning contains viable, possible
pathogenic micro- organisms).

3.11 Instructions of UNESCO-IHE personnel should be obeyed.


CONTENTS

Chapter Page
__________________________________________________________
1. SAMPLING................................................................................................................. 5

2. DIGESTIONS ............................................................................................................. 9
2.1 Digestion of plant samples with H2SO4/Se/salicylic acid and H2O2 ......................... 9
2.2 The destruction of plants and fish tissue for the determination of Cd, Cr, Cu, Pb,
Mn, Fe and Zn with the atomic absorption technique ............................................... 11
2.3 The destruction of soil and sludge for the determination of Cd, Cr, Cu, Pb, Mn,
Fe and Zn with the atomic absorption technique ...................................................... 12
2.4 Preliminary Digestion for Metals in water ................................................................. 14
2.5 Digestion of water samples with the macro-Kjeldahl method ................................... 16
2.6 Digestion of water samples with the persulfate method, acc. to Koroleff ................. 18
2.7 Digestion of water samples for the determination of P ............................................. 20

3. DETERMINATION OF CATIONS............................................................................. 21
3.1 Al; eriochrome cyanine R method ............................................................................ 21
3.2 Ammonia .................................................................................................................. 24
3.3 Ca; EDTA Titrimetric Method.................................................................................... 26
3.4 Ca + Mg (Hardness); EDTA Titrimetric Method........................................................ 27
3.5 Fe; phenanthroline method....................................................................................... 30
3.6 K; flame emission photometry .................................................................................. 34
3.7 Na; flame emission photometry ................................................................................ 35

4. DETERMINATION OF ANIONS............................................................................... 37
4.1 HCO3- / CO32- ; Alkalinity; Titrimetric analysis ........................................................... 37
4.2 Cl-; argentometric titration ........................................................................................ 39
4.3 NO3 ; Procedures...................................................................................................... 41
4.3.1 NO3-; Spectrophotomretric method with 2,6-dimethylphenol ................................... 41
4.3.2 NO3-; Cadmium reduction spectrophotometric method ........................................... 43
4.3.3 NO3-; U.V. spectrophotometric screening method................................................... 46
4.4 NO2-, spectrophotometric method ........................................................................... 48
4.5 PO43-; ascorbic acid spectrophotometric method.................................................... 50
4.6 SO42-; turbidimetric method..................................................................................... 53

5. OXYGEN .................................................................................................................. 55
5.1 Dissolved Oxygen (DO); Azide modification............................................................. 55
5.2 Biochemical Oxygen Demand (BOD) ....................................................................... 57
5.3 Chemical Oxygen Demand (COD) ........................................................................... 67
5.4 Total Organic Carbon (TOC) .................................................................................... 72

6. MISCELLANEOUS .................................................................................................. 75
6.1 Biomass.................................................................................................................... 75
6.2 Suspended Solids, (SS or dry weight)
and Volatile Suspended Solids (VSS or ash-free dry weight)................................... 78
6.3 Chlorophyll-a in algae............................................................................................... 79
6.3.7 Chlorophyll-a in macrophytes ................................................................................... 81
CONTENTS

Chapter Page
__________________________________________________________

6.4 Polycyclic Aromatic Hydrocarbons (PAH) ................................................................ 83


6.5 Cation Exchange Capacity of soil (CEC).................................................................. 85
6.6 Determination of anion-active detergents................................................................. 87
6.7 Boron; azomethine-H method................................................................................... 89
6.8 SiO2; molybdosilicate method................................................................................... 91
6.9 Selective extraction procedure for As(III) ................................................................. 93
6.10 Colorimetric Method for determination of Chromium (VI) ......................................... 95
6.11 Determination of CaCO3 in sediment ....................................................................... 98
6.12 Acid Volatile Sulphide, AVS, in sediment Colorimetric Method .............................. 99
Sampling

1. Sampling

Sample containers

Polyethylene (p.e) or glass bottles are often used; in many cases both materials are equally
satisfactory. The ability to seal the bottles tightly with stoppers or caps is important.
Advantage of glass: can be more vigorously cleaned e.g. sterilized by heating for
bacteriological samples.
Disadvantage: breakage.

Analytical methods often recommend the container type, however different publications do
not always agree. In this manual the guidelines according to ISO 5667-3 about preservation
and handlings of water samples are given.

Considerations in choosing containers

a) The material of the containers may cause contamination of samples, e.g. sodium and
silica can be leached from glass, organic substances can be leached from plastics
b) Determinants may be sorbed on the walls of containers, e.g. trace metals by ion-
exchange processes on glass surfaces, adsorption of benzene by plastics
c) Constituents of the sample may react with the container material e.g. fluoride may react
with glass.

These effects become more and more important as the concentrations of determinants
become smaller.

In general: use glass for determination of organic materials and p.e. bottles should be
used for determinants that are major constituents of glass, e.g. Na+, K+,
boron and silica.

Not only the major components of the container can cause contamination, errors have been
reported from Fe, Mn, Zn and Pb leached from glass and from Li and Cu leached from p.e.

Clean glass containers with chromic acid and p.e. containers with 1 M HCl.
Avoid the use of concentrated HNO3 for p.e. containers since this acid can form chemical
groups with ion-exchange properties.
Don’t use household detergents (PO4!).
Be careful with the use of bottle caps with inserts.

Sample containers may also cause errors by sorption of determinants. Trace metals, e.g. Hg
and Ag, detergents, pesticides and PO4 can easely be absorbed.

This degree of sorption depends on many factors, the addition of suitable preserving
reagents is the best way to avoid problems. Make experimental checks to ensure that
sorption effects are tolerably small.

5
Sampling

Sample storage
Many chemical, biological and physical reactions may occur during this period.
Precautions must be taken otherwise the samples on analysis may be quite
unrepresentative of conditions at the time of sampling.

Some examples of possible reactions:

a) bacteria, algae and other biological species may consume, excrete, or change the
chemical form of many determinants e.g. D.O., BOD, CO2, hardness, alkalinity, pH, NO3,
NH4, PO4
b) determinants may be oxidized by D.O or air e.g. Fe2+, S2-
c) substances may precipitate e.g. CaCO3, metals

d) pH, conductivity, alkalinity, CO2, hardness may change due to CO2 absorption from the
air
e) dissolved and colloidal metals and certain organic components may be absorbed on the
surface of sample containers or solids in the samples
f) polymeric materials may depolymerize condensed inorganic phosphates and polymeric
silicic acid.

Such reactions are generally affected by the chemical and biological nature of the sample,
by its temperature and degree of exposure to light, and by the nature of the sample
container.
Thus, the rate of change of a given determinant may vary not only from one type of water to
another but also from time to time for samples from a given body of water! It is emphasized
that these rates of deterioration are such that important changes sometimes occur within a
few hours or less, and often within a day.

A. Special storage of samples

Biological activity is usually prevented or markedly reduced by storing samples in the dark
and at low temperature, e.g., approximately 4ºC.
This method of preservation is widely employed, and can be very useful; it is always worth
adopting as a minimum precaution when there is doubt about stability. Some samples may
be so unstable as to require refrigeration immediately after collection.
Some researchers recommend storage of sea water samples in p.e. bottles in deep-freeze
units (-20ºC) until just before analysis. They claim that adequate stability for many weeks is
then obtained for determinants such as phosphorus, nitrogen and silicon compounds. The
technique has also been recommended for sewage effluents, even BOD may be reasonably
preserved, and for estuarine waters and fresh waters. The technique has been mainly used
for non-metallic determinants, but has been reported to be satisfactory for mercury in sea-
water. Other researchers, however, have reported errors caused by deep-freezing when
silicon or phosphate are to be determined.

B. Immediate treatment of the sample

Algal and bacterial activity in the sample can often be sufficiently reduced by filtering the
sample through a filter of small pore-size which retains algae and bacteria. Membrane filters
(0.45 μm) or glass-filbre filters are often used for this purpose. The technique is quite
convenient and recommended for phosphorus and silicon compounds. An important
precaution is to ensure that impurities leached from the filter do not cause
appreciable contamination; extensive prewashing of the filter is sometimes needed to
prevent this effect.

6
Sampling

Another possible approach is to begin the analysis of the sample directly after collection so
that the determinant is converted to a form in which it is adequately stable. The analysis can
then be completed when convenient.

The determination of dissolved oxygen by the Winkler method can be used in this way;
addition of the mangenese and alkaline reagents leads to the formation of the oxidized
manganese precipitate, which is stable for relatively long periods. This approach is not
generally applicable, however.

C. Use of special sample containers

It may be virtually impossible to prevent the sorption of certain determinants on the walls of
sample containers, e.g., oils and greases, organochlorine pesticides at very low
concentrations. In such instances, it is necessary to use one sample container solely for a
particular determinant. This allows the sorbed material to be removed from the bottle (e.g. by
solvent extraction) after the sample has been poured into the vessel used for the analysis.

D. Addition of a preserving reagent

Satisfactory stability of many determinants can often be achieved by adding a chemical


reagent to the sample directly after collection. Many reagents have been used, and different
workers often use different concentrations of the same reagent. In addition, the conclusions
reached by authors on the relative suitabilities of different reagents do not always agree.
Nevertheless, the use of preserving reagents can be an extremely useful and relatively
convenient approach, particularly when one reagent stabilizes a number of determinants.

There is general agreement that acidification of samples is necessary when trace metals are
to be determined, but different acids and acidities are recommended. The minimum acidity
required for stability depends on the metal, but there appears to be a growing tendency to
use sufficient nitric or hydrochloric acids to give concentrations in the range 0.05-0.1 mol/L in
the sample after collection; 0.1-0.3 mol/L appears to be essential when mercury and silver
are to be determined.

Biocidal agents are often used when determinants liable to biological reactions are of
interest, e.g. certain organic compounds, nitrogen and phosphorus compounds. Chloroform
has been extensively used in the past, but the modern tendency is to avoid this reagent, and
mercuric chloride or acidification are more frequently recommended. In general, mercuric
chloride (20-40 mg/L) seems to be preferred for preserving nitrogen compounds, while
sulfuric acid (1-2 ml/L) is used for determinants such as COD, fats and greases; no general
conclusions for phosphorus seem justified. However, it was reported that mercuric chloride
in a sample concentration of 40 mg/L did not inhibit microbiological growth in wastewater
effluent samples containing TOC concentrations greater than 20 mg/L, addition of 400 mg/L
was good enough where preservation of nitrogen and phosphorus compounds is desired.
Care is required because determinants such as orthophosphate are released when species
such as algae are killed.

7
Sampling

Types of preservation suitable for different determinants, acc. to ISO 5667-3

Determinant Material of Method of preservation Maximum time between


sample sampling and analysis
container a
Ca, Mg, Na, K P, acid washed HNO3, pH 1-2 1 month
Anions (Br, F, Cl, P or G Cool to 1-5ºC or freeze -20ºC 24 h. respectively 1 month.
NO2, NO3, PO4, SO4) Filter on-site
Conductivity P or BG Cool to 1- 5ºC 24 h. Exclude air
Fluoride P, not PTFE None needed 1 month
Manganese P/BG acid washed HNO3, pH 1-2 1 month
Sulfate P or G Cool to 1- 5ºC 1 month
Alkalinity P or G Cool to 1- 5ºC 24 hours
BOD P or G Cool to 1- 5ºC or freeze to -20ºC 24 h. resp. 1 month. in the dark,
exclude air
Colour P or G Cool to 1- 5ºC 5 days, in the dark.
Nitrogen, ammonia P or G H2SO4, pH 1-2 and cooling 1-5ºC 21 days. Filter on-site
Nitrogen, nitrate P or G HCl, pH 1-2 or cool to 1-5ºC or freeze 7 days resp. 24 h resp. 1 month
–20ºC
Nitrogen, nitrite P or G Cool to 1- 5ºC 2 days. Preferably on-site
measurement
N, Kjeldahl and TN P H2SO4, pH 1-2 or cooling -20ºC 6 months in dark, TN: 1 month
Odour G Cool to 1- 5ºC 6h
Phosphorus, total P/G/BG acid H2SO4, pH 1-2 or freeze -20ºC 6 months
washed
Phosphorus, P/G/BG acid Cool to 1- 5ºC or freeze to -20ºC 1 month. Filter on-site
dissolved washed
Silicates P Cool to 1- 5ºC 1 month
COD P or G Cool to 1- 5ºC or freeze to -20ºC 24 h. resp. 6 month. in the dark
Oil/grease G solvent washed H2SO4, or HCl, pH 1-2 1 month
TOC P or G H2SO4, pH 1-2 and cool 1- 5 ºC 7 days respectively 1 month
or just freezing to - 20 ºC
Iron (II) P/BG acid washed HCl, pH 1-2, fill container completely 7 days
Iron, total P/BG acid washed HNO3, pH 1-2 1 month
Heavy metals, P/BG acid washed HNO , pH 1-2 6 months
3
except for mercury
Mercury BG acid washed HNO3, pH 1-2 , add K2Cr2O7 1 month
until final conc of 0.05%
Cyanide, total P NaOH, pH > 12 and cool 1-5 ºC 24 h – 7 days, depends on S2-
Sulfide P Add 1mol.l-1 Zn (OAc)2, 2 mL per litre of 7 days
sample. Fill container completely. Add 80
mg ascorbic acid / 100 mL chlorinated
sample prior to analysis
Chlorine P or G Keep in the dark for max. 5 min 5 min
Dissolved oxygen P or G Fill container completely Analyse as soon as possible,
preferably on site
pH P or G Cool to 1- 5ºC, fill container completely 6 h. Analyse as soon as
possible, preferably on site
Solids, TSS P or G Cool to 1- 5ºC 2 days
Solids, TDS P or G Cool to 1- 5ºC 24 h
Sulfite P or G Add 1 mL 2.5% EDTA /100 mL 2 days
Turbidity P or G Cool to 1- 5ºC 24 h, in the dark

a P = polyethylene; G = glass
d The maximum time depends on the type of sample

Literature:
Examination of water for pollution control
Volume 1 Sampling, data analysis and laboratory equipment
J. Suess ISBN 0-08-025255-9

8
Digestions

2. DIGESTIONS

2.1 Digestion of plant samples with H2SO4/Se/salicylic acid and H2O2

2.1.1 Field of application

This digestion procedure can be applied for the determination of N-total (finally measured as
NH4), P, Na, K, Ca, Mg and Zn.
The Ca content of the plant samples should not exceed 45 g Ca per kg dry weight.

2.1.2 Principle

The large part of organic matter is oxidized by H2O2 at relatively low temperature. After
decomposition of the excess H2O2 and evaporation of water, the digestion is completed by
conc. H2O4 at approximately 300°C under the influence of Se as a catalyst.
Salicylic acid is used to form nitro-salicylic acid compounds in order to prevent loss of free
nitrate.
Since CaSO4 may be formed when cooling after completing the digestion it is necessary to
wait 24 hours after the addition of water before Ca analysis. During this period the CaSO4
will dissolve.

2.1.3 Apparatus

Heating block, for temperatures up to 300°C, with holes for tubes.


Destruction tubes made of glass, acid rinsed and dried.

2.1.4 Reagents, all p.a. grade

a) Conc. H2SO4
b) H2O2 30%
c) Se powder
d) Salicylic acid, powder
e) Preparation of H2SO4 - Se mixture
Heat 250 mL of conc. H2SO4 on a hot plate until fumes will appear (± 300°C), add
0.88 g Se while mixing. Keep the temperature high. The originally dark coloured
suspension turns via green-blue into a clear yellowish solution. Cool down.
f) Digestion mixture
Dissolve 7.2 g of salicylic acid in 100 mL H2SO4 - Se mixture at room temperature. The
colour will change from blue to yellow/greenish while mixing. This solution should not be
stored for more than 48 hours.

2.1.5 Procedure

1. Weight about 0.3 g with an accuracy of 0.001 g of the dried plant material and transfer
quantitatively to the destruction tube (do not weigh less than 0.1 g)
2. Add 2.5 mL digestion mixture, swirl carefully until all the plant material is moistened.
Prepare also 2 blanks and 2 reference samples
3. Allow standing for at least 2 hours, during these period nitro-salicylic acid compounds will
be formed

9
Digestions

4. Place the tube at 100°C for at least 2 hours in an oven, during this period the nitro-
salicylic compounds will be reduced
5. Cool the tubes to room temperature and add successively three 1 mL aliquots of H2O2,
mix carefully after each addition. The reaction is violent, wait until the reaction with H2O2
has ceased (±10 sec) before adding the next portion
6. Place the tube again in the preheated block and heat at 330°C. In the beginning the rack
with tubes should be lifted up from time to time to prevent loss of liquid during boiling. The
digestion is considered complete when the digests have turned colourless or light yellow;
this usually takes about 2 hours
7. The digest is diluted with about 15 mL of water, add about five pumice grains, boil (lower
the rack with tubes a few cm in the heating block, be careful!) and after cooling made up
to 50 mL in a volumetric flask. Mix well, let particles settle for 24 hours, before analysis
8. Continue as described in the Procedure for the ammonia determination and use a
portion of the neutralized digested sample containing not more than 0.1 mg N

Remarks:
1) Before weighing, the plant material should contain a moisture content of 1-2%, so store
the plant material in a dessicator after drying or dry again at 70°C before weighing. If this
is not done, the plant sample may contain up to 10% moisture. The use of conc. H2SO4
then causes a raise in temperature, which will result in a loss of nitrate.

2) The plant sample is normally dried at 70°C in a well-ventilated drying oven during
24 hours. The material is then finely ground, in order to obtain a homogeneous sample
from which representative subsamples can simply be taken. As a rule of thumb, the
milled plant material should pass a 1 mm sieve when less than 1 gram is to be weighed
out. Both drying and milling should be carried out with equipment that does not release
elements for which the samples are to be analysed.
The dried and milled samples should be stored in a cool and dry place in tightly
stoppered flasks or in sealed polyethene bags, protected against direct sunlight. During
storage, the plant material may attract moisture so that the drying procedure at 70ºC
must be repeated just before weighing out a sample for analysis.
The analytical results are often referred to “oven-dry” material, which means dried
at 105ºC. For comparability, therefore, the moisture content should be determined
by drying at 105ºC and taking the difference with the 70ºC dried sample. The drying
at 105ºC should be done, however, with a separate sample, since this operation
may change its chemical composition.

Literature:
Syllabi Soil and Plant analysis, part 7, plant analysis procedures.

Dept. of Soil Science and Plant Nutrition by Walinga, Vark, Houba, v.d. Lee 1989
Wageningen Agriculture University.

10
Digestions

2.2 The destruction of plants and fish tissue for the determination of Cd,
Cr, Cu, Pb, Mn, Fe and Zn with the atomic absorption technique

2.2.1 Apparatus

a) Destruction-bloc with destruction tubes made of borosilicate glass


b) Nichiryo pipet model 3100 with removable tips

2.2.2 Reagents, all with a low metal content

a) Nitric acid, 65% HNO3


b) Hydrogen peroxide, 30% H2O2
c) Pumice

2.2.3 Glassware

All rinsed with 1 + 1 HNO3


Measuring cylinder, 50 mL
Funnels with a diameter of 6 cm
Volumetric flasks of 250 mL

2.2.4 Procedure

1. Transfer not more than 1.250 g dried sample (24 hours at 103°C) to the destruction tube,
add 25 mL HNO3, three boiling chips and place a funnel on top of the destruction tube.

2. a) Heat the tube to 100°C and maintain for 1 hour


b) Heat to 125°C and maintain for 15 minutes
c) Heat to 150°C and maintain for 15 minutes
d) Heat to 175°C and maintain for 15 minutes
e) Heat to 200°C and add, if necessary (if no volume is left), 5 mL HNO3
3. Concentrate to about 5 mL
4. Add, after cooling, 1 mL 30% H2O2 and destruct for 10 minutes, repeat 1 x
5. Add, after cooling, 3 mL 30% H2O2 and destruct again for 10 minutes
6. Add 25 mL water, mix and heat till boiling
7. Cool and transfer the whole sample to a 250 mL volumetric flask, fill up to the mark, mix
and let settle during at least 15 hours
8. Measure the absorbance of the clear supernatant.

Note: 1. Duration of procedure steps 1-7 at least 7 hours


2. Determine two blancs

11
Digestions

2.3 The destruction of soil and sludge for the determination of Cd, Cr, Cu,
Pb, Mn, Fe and Zn with the atomic absorption technique

2.3.1 Apparatus

a) Destruction-bloc with destruction tubes made of borosilicate glass


b) Nichiryo pipet model 3100 with removable tips

2.3.2 Reagents

All reagents with a low percentage of heavy metals


a) Hydrochloric acid, 37% HCl
b) Nitric acid, 65% HNO3
c) Hydrogen peroxide, 30% H2O2
d) Pumice

2.3.3 Glassware

All rinsed with 1 + 1 HNO3


Measuring cylinder, 500 mL
Measuring cylinder, 50 mL
Funnels with a diameter of 6 cm
1 L flask for the acid-mixure, see note
Volumetric flasks of 250 mL

2.3.4 Procedure

1. Transfer not more than 1.250 g of a ground air-dried sample to the destruction tube, add
50 mL H2O and three boiling chips
2. Add 50 mL HCl/HNO3 3:1, mix, and place a funnel on top of the destruction tube

3. a) Heat the tube to 100°C and maintain for 1 hour


b) Heat to 125°C and maintain for 15 minutes
c) Heat to 150°C and maintain for 15 minutes
d) Heat to 175°C and maintain for 15 minutes
e) Heat to 200°C and add, if necessary (if no volume is left), 5 mL HNO3
4. Concentrate to about 5 mL
5. Add, after cooling, 1 mL 30% H2O2 and destruct for 10 minutes. Repeat 1 x
6. Add, after cooling, 3 mL 30% H2O2 and destruct again for 10 minutes
7. Add 50 mL water and 25 mL HCl, mix and heat till boiling
8. Cool and transfer the whole sample to a 250 mL volumetric flask, fill up to the mark, mix,
and let settle during at least 15 hours
9. Measure the absorbance of the clear supernatant.

12
Digestions

Note: 1. Prepare a fresh acid-mixture and do not close the container!!


2. Duration of procedure steps 1-7 at least 7 hours
3. Determine two blancs
4. Sludge samples will decompose almost completely

Literature:

1. NEN 6465 (Dutch Standards). Sample pretreatment of sludge, sludge containing water
and air-dust for the determination of elements with atomic absorption spectrometry.
Destruction with nitric acid and hydrochloric acid

2. Atomic Absorption News letter Vol 17, no. 4


Comparison of sample preparation techniques for atomic absorption analysis of sewage
sludge and soil

3. P. Schramel, Li-Qiang, A. Wolff, S. Hasse, 1982


ICP-Emissionsspektroskopie:
Ein analytisches Verfahren zur Klärschlamm- und Bodenüberwachung in der Routine.
Fresenius Z. Anal. Chem. (1982) 313: 213-216.

13
Digestions

2.4 Preliminary Digestion for Metals in water

To reduce interference by organic matter and to convert metal associated with particulates
to a form (usually the free metal) that can be determined by atomic absorption spectrometry
or inductively-coupled plasma spectroscopy, use one of the digestion techniques presented
below. Use the least rigorous digestion method required to provide complete and consistent
recovery compatible with the analytical method and the metal being analyzed.

Nitric acid will digest most samples adequately. Nitrate is an acceptable matrix for both flame
and electrothermal atomic absorption. Some samples may require addition of perchloric,
hydrochloric, or sulfuric acid for complete digestion. These acids may interfere in the
analysis of some metals and all provide a poorer matrix for electrothermal analysis. Confirm
metal recovery for each digestion and analytical procedure used. As a general rule, HNO3
alone is adequate for clean samples or easily oxidized materials: HNO3-H2SO4 or HNO3-HCl
digestion is adequate for readily oxidizable organic matter; HNO3-HClO4 or HNO3-HClO4-HF
digestion is necessary for difficult-to-oxidize organic matter or minerals. Dry ashing is helpful
if large amounts of organic matter are present, but yields highly variable precision and bias,
depending on sample type and metal being analyzed. Dry-ash only samples that have been
shown to yield acceptable precision and bias.

2.4.1 Dry ashing procedure, use only if necessary

Mix sample and tranfser a suitable volume into a platinum or high-silica glass evaporating
dish. Evaporate to dryness on a steam bath. Transfer dish to a muffle furnace and heat
sample to a white ash. If volatile elements are to be determined, keep temperature at 400 to
450°C. If sodium only is to be determined, ash sample at a temperature up to 600°C.
Dissolve ash in a minimum quantity of conc HNO3 and warm water. Filter diluted sample and
adjust to a known volume, preferably so that the final HNO3 concentration is about 1%. Take
portions of this solution for metals determination.

2.4.2 Nitric acid digestion procedure

Apparatus

a) Hot plate
b) Conical flasks, 100 mL, acid-washed and rinsed with water

Reagents

Nitric acid, HNO3, conc.

Procedure

Mix sample and transfer a suitable volume (50 to 100 mL) to a 100 mL conical flask or
beaker. Add 5 mL conc. HNO3 and a few boiling chips. Bring to a slow boil and evaporate on
a hot plate to the lowest volume possible (about 10 to 20 mL) before precipitation occurs.
Continue heating and adding conc HNO3 as necessary until digestion is complete as shown
by a light-colored, clear solution. Do not let sample dry during digestion.
Wash down flask or beaker walls with water and then filter if necessary. Transfer filtrate to a
100 mL volumetric flask with two 5 mL portions of water, adding these rinsings to the
volumetric flask. Cool. dilute to mark, and mix thoroughly. Take portions of this solution for
required metal determinations.

14
Digestions

2.4.3 Nitric Acid-Hydrochloric Acid Digestion

Apparatus see 2.4.2

Reagents

a) Nitric acid, HNO3, conc.


b) Hydrochloric acid, 1 + 1

Procedure

a) Total HNO3/HCl: Transfer a measured volume of wellmixed, acid-preserved sample


appropriate for the expected metals concentrations to a flask or beaker. Add 3 mL conc
HNO3. Place flask or beaker on a hot plate and cautiously evaporate to less than 5 mL ,
making certain that sample does not boil and that no area of the bottom of the container
is allowed to go dry. Cool and add 5 mL conc HNO3. Cover container with a watch glass
and return to hot plate. Increase temperature of hot plate so that a gentle reflux action
occurs. Continue heating, adding additional acid as necessary, until digestion is complete
(generally indicated when the digestate is light in color or does not change in appearance
with continued refluxing). Evaporate to less than 5 mL and cool. Add 10 mL 1 + 1 HCl
and 15 mL water per 100 mL anticipated final volume. Heat for an additional 15 min to
dissolve any precipitate or residue. Cool, wash down beaker walls and watch glass with
water, and adjust to a predetermined volume based on the expected metals
concentrations. If necessary (when particles are present), let settle overnight.

b) Recoverable HNO3/HCl: For this less rigorous digestion procedure, transfer a measured
volume of well-mixed, acid-preserved sample to a flask or beaker. Add 2 mL 1 + 1 HNO3
and 10 mL 1 + 1 HCl and heat on a steam bath or hot plate until volume has been
reduced to near 25 mL, making certain sample does not boil. Cool and let settle
overnight. Adjust volume to 100 mL and mix.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 3-4/3-7.

15
Digestions

2.5 Digestion of water samples with the macro-Kjeldahl method

2.5.1 Field of application

The macro-Kjeldahl method is applicable for samples containing either low or high
concentrations of organic nitrogen but requires a relatively large sample volume for low
concentrations.

2.5.2 Storage of samples

The most reliable results are obtained on fresh samples. If an immediate analysis is not
possible, preserve samples by acidifying to pH 1.5-2.0 with conc. H2SO4 and storing at 4°C.
Do not use HgCl2 because it will interfere with ammonia removal.

2.5.3 Interferences

During digestion, nitrate in excess of 10 mg/L can oxidize a portion of the ammonia released
from the digested organic nitrogen, producing N2O and resulting in a negative interference.
When sufficient organic matter in a low state of oxidation is present, nitrate can be reduced
to ammonia, resulting in a positive interference.

2.5.4 General discussion

Nitrogen containing compounds will be converted to NH4+.


Nitrogen in the form of azide, azine, azo, hydrazone, nitrate, nitrite, nitrile, nitro, oxime and
semi-carbazone is not included. If ammonia is not removed in the initial phase the term
"Kjeldahl-nitrogen" is applied to the result, if ammonia is removed the term "organic nitrogen"
is applied.

2.5.5 Principle

In the presence of H2SO4, K2SO4 and CuSO4 catalyst, amino nitrogen of many organic
materials is converted to (NH4)2SO4. Also NH3 and NH4+ are converted to (NH4)2SO4. During
sample digestion, a copper ammonium complex is formed and afterwards decomposed by
Na2S2O3. After decomposition the NH3 is distilled from an alkaline medium and absorbed in
H2SO4.

2.5.6 Apparatus

Digestion apparatus
Distillation apparatus

2.5.7 Reagents

a) Digestion reagent: Dissolve 134 g K2SO4 and 11.4 g CuSO4.5H2O in about 800 mL H2O.
Carefully add 134 mL conc. H2SO4. When it has cooled to room temperature dilute to 1 L.
Keep at a temperature close to 20°C to prevent crystallization
b) NaOH - Na2S2O3 reagent: Dissolve 500 g NaOH and 25 g Na2S2O3.5 H2O in water and
dilute to 1 L
c) H2SO4 0.02 M: Add 1.2 mL conc. H2SO4 to 1 L H2O
d) Intermediate standard NH4Cl for Kjeldahl determination:

16
Digestions

Dilute 50.00 mL stock solution to 1000 mL 1.00 mL = 0.05 mg N


2.5.8 Procedure

1. Place a measured volume of sample and blank in a Kjeldahl flask, select sample size
from the following tabulation. If necessary, add water till about 300 mL. Neutralize to
pH 7 in case of strong alkaline samples.

Expected N mg/L in sample Sample size, mL


0 - 1 500
1 - 10 250
10 - 20 100
20 - 50 50
50 - 100 25
100 - 200 10
200 - 250 5

2. Carefully add 50 mL digestion reagent, mix well and add a few pumice grains
3. Heat in the hood and boil until the volume is about 25 mL and fumes are observed
4. Continue to digest for an additional 30 min., coloured or turbid samples will turn clear or
straw-coloured. The total destruction time must be at least 4 hours.
5. Cool, dilute to 300 mL and mix
6. Carefully add 50 mL NaOH-Na2S2O3 mixture and connect immediately the flask to the
distillation apparatus. Shake the flask to insure complete mixing. A black HgS
precipitate will form, the pH should exceed 11.0
7. Distil and collect 200 mL distillate below the surface of 50 mL 0.02 M H2SO4. Extend tip
of condenser well below level of H2SO4 and do not let temperature in condenser rise
above 29°C.
Use a medium high flame and do not lower this flame during the distillation.
At the end of the distillation, remove the distillate before the flame is turned off
8. Measure the end volume by means of a measuring cylinder and mix
9. Determine N content of the sample, see procedure 3.2 and start with procedure 3.2.6
step 3
10. Distillation recovery check: Treat a standard in the same way as the sample by putting
25.00 mL of the intermediate standard solution (= 1.25 mg N) in the Kjeldahl flask as
described in step 1
Measure the end volume by means of a measuring cylinder and mix. Take 10.00 mL
distillate and measure the absorbance against mg N in 50 mL (see procedure 3.2.6,
starting at step 3). Check the distillation recovery

Remark:
If ammonia should be removed: add 25 mL borate buffer (9.5 gram Na2B4O7.10H2O/800 mL
+ 200 mL 0.1 M NaOH) and 6 M NaOH until pH 9.5 is reached.
Add some pumice and boil off 300 mL. If desired, distill the fraction and determine ammonia
nitrogen. Use the residue to determine organic nitrogen.

Literature:
American Public Health Organisation. Standard Methods for the examiniation of water and
wastewater, 18th edition 1992. p. 4-95/4-97.

17
Digestions

2.6 Digestion of water samples with the persulfate method, acc. to Koroleff

2.6.1 Field of application

The persulfate method determines total nitrogen by oxidation of all nitrogenous compounds
to nitrate. The organic content in the water samples should not be too high. Should
ammonia, nitrate and nitrite be determined individually, “organic nitrogen” can be obtained
by the difference.The procedure gives good results for total nitrogen, composed of organic
nitrogen (including some aromatic nitrogen-containing compounds), ammonia, nitrate and
nitrite. Molecular nitrogen is not determined and recovery of some industrial nitrogen-
containing compounds is low.

2.6.2 Storage of samples

Samples may not be preserved with acid!


Preserve samples by cooling.

2.6.3 Interferences

Chloride ions do not interfere with persulfate oxidation, but the rate of reduction of nitrate to
nitrite (during subsequent nitrate analysis by the Cd-reduction method) is significantly
decreased by chlorides. Ammonium and nitrate ions adsorbed on suspended pure clay or
silt particles should give a quantitative yield from persulfate digestion. If suspended matter
remains after digestion, remove it before the Cd-reduction step.
If suspended organic matter is dissolved by the persulfate digestion reagent, yields
comparable to those from true solutions are obtained; if it is not dissolved, the results are
unreliable and probably reflect a negative interference.
The persulfate is not effective in wastes with high organic loadings, dilute such
samples and re-analyze until results from two dilutions agree.

2.6.4 Principle

Alkaline oxidation with persulfate at 100 – 110 °C converts organic and inorganic nitrogen to
nitrate. Total nitrogen is determined by analyzing the nitrate in the digestate.

2.6.5 Apparatus

Hot plate, autoclave or microwave

2.6.6 Reagents

a) Dissolve 15 g NaOH in 1000 mL H2O


b) Dissolve 5.0 g K2S2O8, potassiumperoxodisulphate, in 80 mL of solution a)
This will take some time.
Add 3.0 g H3BO3, boric acid, now everything will dissolve easily.
Fill up to 100 mL with solution a), use a volumetric flask. pH=12
This solution is stable for 1 week
c) Glycine, the digestion check standard
Dissolve 1.072 g glycine in 1000 mL H2O; 1.00 mL = 200 μg N
This stock solution can be stored during 1 month at 4 °C

18
Digestions

d) Intermediate glycine digestion standard


Dilute 5.00 mL of solution c) to 250 mL with H2O; 1.00 mL = 4.00 μg N
Prepare this solution fresh on the day of use
e) Nitrate standard solution; 1.00 mL = 10.0 μg NO3-N. See 4.3.2.5

2.6.7 Calibration

1. Transfer 0; 5.00; 10.00; 15.00; 20.00 and 25.00 mL standard nitrate solution in 100 mL
erlenmeyer flasks. The amount of N is in the range of 0-250 μg N. Fix a paper with a
description of the sample/standard around the neck of the erlenmeyer flask, use a
pencil to write down the information on the label
2. Add 5.0 mL solution b) and bring all volumes to about 30 mL with H2O
3. Cover the erlenmeyer flask with a cotton plug and aluminium foil and mix carefully
4. Put the flasks in the autoclave and destruct for 30 min. at 110 °C
5. Cool the flasks and transfer the contents to 50.0 mL volumetric flasks and mix
6. Measure the absorbance at 220 and 275 nm as described in procedure 4.3.3

2.6.8 Procedure for the sample

According to the calibration procedure, take a sample volume which contains less than
500 μg N, this is the maximum amount which may be present in 50 mL end-volume.
Check each digestion procedure: transfer 25.00 mL intermediate glycine digestion standard
to a 100 mL erlenmeyer flask and treat this standard in the same way as the samples.

2.6.9 Remark

The glassware to be used for the digestion should be rinsed before use, do this by filling the
erlenmeyers with solution b) and treat them in the autoclave by following the above
mentioned procedure.

Literature:
American Public Health Organisation
Standard Methods for the examination of water and wastewater, 20 th edition 1998
p. 4-99 / 4-103

UNEO / WHO / UNESCO / WMO


Project on Global Monitoring GEMS / Water Operational Guide, Chapter III: Analytical Guide,
Copenhagen, Denmark

19
Digestions

2.7 Digestion of water samples for the determination of P

2.7.1 Introduction

Phosphorus occurs in natural waters and in wastewaters almost solely as phosphates.


These are classified as orthophosphates, condensed phosphates (pyro-, meta-, and other
polyphosphates), and organically bound phosphates. They occur in solution, in particles or
detritus, or in the bodies of aquatic organisms.
Phosphates also occur in bottom sediments and in biological sludges, both as precipitated
inorganic forms and incorporated into organic compounds.

Phosphorus analyses embody two general procedural steps:


a. conversion of the phosphorus form of interest to dissolved orthophosphate, and
b. colorimetric determination of dissolved orthophosphate.

2.7.2 Sampling and storage

If phosphorus forms are to be differentiated, filter sample immediately after collection.


Preserve by freezing at or below -10ºC. Add 40 mg HgCl2/L to the samples, especially when
they are to be stored for long periods.
Caution: HgCl2 is a hazardous substance; take appropriate precautions in disposal. Do not
add either acid or CHCl3 as a preservative when phosphorus forms are to be determined. If
total phosphorus alone is to be determined, add 1 mL conc. HCl/L or freeze without any
additions.

2.7.3 Apparatus and glassware

Hot plate placed in the hood


GF/C filters
Acid-rinsed 50 mL erlenmeyer flasks and acid-rinsed 100 mL volumetric flasks

2.7.4 Reagents

a) conc. H2SO4 ; conc. HNO3 and 6 M NaOH


b) Phenolphthaleine indicator: Dissolve 0.5 g in 50 mL ethanol and add 50 mL H2O

2.7.5 Procedure

1) Transfer a volume of sample, standard or blank (each containing not more than 0.1 mg
P) to the erlenmeyer flask and add 1 mL conc. H2SO4, 5 mL conc. HNO3 and some
pumice
2) Digest to a volume of 1 mL and than continue until solution becomes colourless to
remove HNO3
3) Cool and add approximately 20 mL H2O, 1 drop indicator and as much 6 M NaOH as
required to produce a faint pink tinge
4) If necessary, remove particulate material or turbidity by filtering and transfer quantitatively
the sample and blank in a 100 mL volumetric flask
5) Measure P as described in procedure 4.5.7; start with step 2.
Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-108/4-110.

20
Determination of cations

3. DETERMINATION OF CATIONS

3.1 Al; eriochrome cyanine R method

3.1.1 Principle

With Eriochrome cyanine R dye, dilute aluminum solutions buffered to a pH of 6.0 produce a
red to pink complex that exhibits maximum absorption at 535 nm. The intensity of the
developed color is influenced by the aluminum concentration, reaction time, temperature,
pH, alkalinity, and concentration of other ions in the sample. To compensate for color and
turbidity, the aluminum in one portion of sample is complexed with EDTA to provide a blank.
The interference of iron and manganese, two elements often found in water, is eliminated by
adding ascorbic acid. The optimum aluminum range lies between 20 and 300 μg/L.

3.1.2 Interference

Negative errors are caused by both fluoride and polyphosphates. When the fluoride
concentration is constant, the percentage error decreases with increasing amounts of
aluminum. Because the fluoride concentration often is known or can be determined readily,
fairly accurate results can be obtained by adding the known amount of fluoride to a set of
standards. A simpler correction can be determined from the family of curves. A procedure is
given for the removal of complex phosphate interference. Orthophosphate in concentrations
under 10 mg/L does not interfere. The interference caused by even small amounts of
alkalinity is removed by acidifying the sample just beyond the neutralization point of methyl
orange. Sulfate does not interfere up to a concentration of 2000 mg/L.

The minimum aluminum concentration detectable by this method in the absence of fluorides
and complex phosphates is approximately 6 μg/L.

3.1.3 Sample handling

Collect samples in clean, acid-rinsed bottles, preferably plastic, and examine them as soon
as possible after collection. If only soluble aluminum is to be determined, filter a portion of
sample through a 0.45 μm membrane filter; discard first 50 mL of filtrate and use succeeding
filtrate for the determination. Do not use filter paper, absorbent cotton, or glass wool for
filtering any solution that is to be tested for aluminum, because they will remove most
of the soluble aluminum.

3.1.4 Apparatus and glassware

a) Spectrophotometer, for use at 535 nm, with a light path of 1 cm or longer


b) Glassware: Treat all glassware with warm 1 + 1 HCl and rinse with aluminum-free
distilled water to avoid errors due to materials absorbed on the glass. Rinse sufficiently to
remove all acid.

3.1.5 Reagents

a) Dissolve 8.791 g aluminum potassium sulfate, AlK(SO4)2.12H2O, in water and dilute to


1000 mL. Correct this weight by using the purity of the Al-salt; 1.00 mL = 500 μg Al
b) Standard aluminum solution: Dilute 2.00 mL stock aluminum solution to 1000 mL with
water; 1.00 mL = 1.00 μg Al. Prepare daily
c) H2SO4, 0.01 M and 3 M (3M H2SO4 : add 83 mL conc. H2SO4 to 400 mL H2O)

21
Determination of cations

d) Ascorbic acid solution: Dissolve 0.1 g ascorbic acid in water and make up to 100 mL in a
volumetric flask. Prepare fresh daily
e) Buffer reagent: Dissolve 136 g sodium acetate, NaC2H3O2.3H2O, in water, add 40 mL 1 M
acetic acid and dilute to 1 L
f) Stock solution Eriochrome cyanine R: Dissolve 300 mg dye in about 50 mL water. Adjust
pH from about 9 to about 2.9 with 1 + 1 acetic acid (approximately 3 mL will be required).
Dilute with water to 100 mL. This stock solution has excellent stability and can be kept for
at least a year.
Working dye reagent: Dilute 10.0 mL stock dye solution to 100 mL in a volumetric flask
with water. Working solutions are stable for at least 6 months
g) Methyl orange indicator solution: Dissolve 50 mg in 100 mL H2O
h) EDTA (sodium salt of ethylenediamine-tetraacetic acid dihydrate), 0.01 M: Dissolve 3.7 g
in water, and dilute to 1 L
i) NaOH 1 M and 0.1 M.

3.1.6 Procedure

1. Prepare a series of aluminium standards from 0 to 20 μg by accurately transferring


0 - 20 mL of standard aluminium solution into 50 mL volumetric flasks. Add water to a
total volume of approximately 25 mL
2. Add 1 mL 0.01 M H2SO4 to each standard and mix
3. Add 1 mL ascorbic acid solution and mix
4. Add 10 mL buffer solution and mix
5. Add 5.00 mL working dye reagent and mix and make up immediately to 50 mL with water.
Mix and let stand for 5 to 10 minutes. The colour can begin to fade after 15 minutes
6. Measure the absorbance at 535 nm (against demi water)
7. Plot absorbance against the concentration of Al (μg Al in 50 mL volumetric flasks).

Sample treatment in absence of fluoride and complex phosphates:


1. Place 25.0 mL sample, or a portion diluted to 25 mL, in a glass beaker, add a few drops
of methyl orange indicator and titrate with 0.01M H2SO4 to a faint pink colour. Record
reading and discard the sample
2. To two similar samples at room temperature add the same amount of 0.01M H2SO4 used
in the titration and 1 mL in excess. Add to one sample 1 mL EDTA, this is the blank (see
remark)
3. To both samples add 1 mL ascorbic acid, 10 mL buffer reagent, and 5.00 mL working dye
reagent. Mix well after each addition, immediately fill up to 50 mL with demi water
4. Set to zero with demi water. Measure the absorbance after 5 to 10 minutes and
determine the aluminium concentration using the calibration curve, compensate for the
sample blank.

Remark, in case of coloured samples and/or turbidity:


To one sample add 1 mL EDTA solution after step 2 in case of colour/turbidity. If there is no
colour/turbidity this step can be skipped. This will serve as a blank by complexing any
aluminium present and compensating for colour and turbidity.

22
Determination of cations

Removal of phosphate interference:

1. Add 1.7 mL 3M H2SO4 to 100 mL sample in a 200 mL erlenmeyer flask. Heat on a hot
plate for at least 90 minutes, keeping solution temperature just below the boiling point. At
the end of the heating period solution volume should be about 25 mL. Add water if
necessary to keep it at or above that volume
2. After cooling, neutralize to a pH of 4.3 to 4.5 with NaOH, using 1 M NaOH at the start and
0.1 M for the final fine adjustment. Monitor with a pH-meter. Make up to 100 mL with
water, mix, and use a 25 mL portion for the aluminum test
3. Run a blank in the same manner, using 100 mL distilled water and 1.7 mL 3 M H2SO4.

Correction for samples containing fluoride:


1. Measure sample fluoride concentration
2. Either: add the same amount of fluoride as in the sample to each aluminum standard or
determine fluoride correction from the set of curves.

3.1.7 Calculation

μg Al ( in 50 mL final volume )
mg Al/L =
mL sample

23
Determination of cations

3.2 Ammonia

3.2.1 Introduction

Direct determination of ammonia can be carried out for drinking waters, clean surface waters
and good-quality nitrified wastewater effluent.
In other instances, and where interferences are present and greater precision is necessary,
a preliminary distillation step is required. This method is proven to be applicable for all
domestic waste waters. Using 40 mL of sample and 50 mm cuvettes a low analytical limit of
0.01 mg/L NH4-N can be achieved.

3.2.2 Interferences

Some amines may interfere with this method.


Destillation as a pretreatment step is necessary when sample is coloured and/or turbid, or
when during colourdevelopment turbidity appears.

3.2.3 Apparatus

a) Spectrophotometer for use at 655 nm with 1 cm cells for samples in the range of 0.05 –
2.0 mg NH4-N/L

b) Waterbath at 25 °C
c) pH meter

3.2.4 Reagents

a) Salicylate reagent: dissolve 130 g sodiumsalicylate (NaC7H5O3), and 130 g


sodiumcitratedihydrat (Na3C6H5O7.2H2O) in about 650 mL demineralized water in a
1000 mL volumetric flask; check the pH not to be higher than 8.0 and add HCl for
adjustment.
Add 0.970 g disodiumpentacyano nitrosylferrate (III) (Na2Fe(CN)5NO.2H2O), mix and fill
up to the mark.
Store in a brown bottle, this reagent can be used for 2 months

b) Dichloroisocyanurate reagent: dissolve 32.0 g NaOH in 500 mL deminerlized water.


Cool down to room temperature and add 2.00 g sodiumdichloroisocyanurate
(NaC3N3O3Cl2). Make up to volume in a 1000 mL volumetric flask and mix. This reagent
is valid for 2 weeks when stored in the refrigerator

c) Stock NH4Cl: Dissolve 3.819 g anhydrous NH4Cl, dried at 105°C, in water and dilute to
1000 mL.
1.00 mL = 1.00 mg N
d) Intermediate standard NH4Cl for Kjeldahl determination:
Dilute 50.00 mL stock solution to 1000 mL
1.00 mL = 0.05 mg N
e) Standard NH4Cl:
Dilute 10.00 mL stock solution to 1000 mL
1.00 mL = 0.01 mg N = 10 μg N
f) Methyl-red indicator: dissolve 100 mg of the sodium salt in 60 mL ethanol and add
40 mL H2O

24
Determination of cations

3.2.5 Calibration

1. Make a series of standards by diluting 0; 0.50; 1.00; 2.00; 3.00; 5.00; 7.00 and 10.00 mL
standard solution (see 3.2.4 e) to 40 mL in a 50 mL volumetric flask
2. Proceed as described in 3.2.6 step 4.
Plot the absorbance against μg NH4-N/50 mL end volume
3. Determine the mathematical expression for the calibration line.

3.2.6 Procedure

1. Bring sample at room temperature and neutralize if necessary.


Neutralize distilled water samples (also Kjeldahl distillates) or digested plant samples with
NaOH and use methyl-red indicator (a 2.00 mL digested plant sample requires about 1-2
mL 2M NaOH)
2. Filter over a glassfiber filter (GF/C) in case of wastewater samples
3. Pipet the sample (maximum of 40 mL) in a 50 mL volumetric flask and dilute to a
maximum of 40 mL
4. Add 4.0 mL salicylate reagent and mix
5. Add 4.0 mL of dichloroisocynurate reagent and mix again (pH should be 12.6 ± 0.1)
6. Fill up to the mark with demi water and mix
7. Determine the absorbance at 655 nm between 1 and 3 hours.

3.2.7 Calculation
μg NH − N in 50 mL endvolume
4
NH4-N mg/L =
mL sample

μg NH − N in 50 mL endvolume
4 mL end volume distillate
Kjeldahl N mg/L = x
mL sample used for destruction mL distillate transferred to 50 mL flask

3.2.8 Remarks

1. The colour development depends on the temperature, place the volumetric flasks of step
3.2.6.5 in a waterbath of 25.0 ± 0.2 °C (if necessary).

Literature:
NEN 6472

25
Determination of cations

3.3 Ca; EDTA Titrimetric Method

3.3.1 Principle

When EDTA is added to water containing both calcium and magnesium, it combines first
with the calcium. Calcium can be determined directly, with EDTA, when the pH is made
sufficiently high that the magnesium is largely precipitated as Mg(OH)2 and an indicator is
used that combines with calcium only.

3.3.2 Interference

Orthophosphate precipitates calcium at the pH of the test. Alkalinity in excess of 300 mg/L
may cause an indistinct end point in hard waters.

3.3.3 Reagents

a) NaOH 1M
b) Murexide indicator: Dissolve 150 mg in 100 g absolute ethylene glycol
c) EDTA titrant 0.01 M: Dissolve 3.723 g Na2 EDTA.2H2O in 1000 mL, standardize this
solution, 1 mmol EDTA = 1 mmol Ca
Restandardize from time to time.

3.3.4 Procedure

1. Use 50.0 mL sample, or a smaller portion diluted to 50 mL so that the calcium content is
about 5-10 mg.
Analyze hard waters with alkalinity higher than 300 mg/L CaCO3 by taking a smaller
portion and diluting to 50 mL or, when the calcium content is low, by neutralizing the
alkalinity with acid, boiling 1 min, and cooling before beginning the procedure

2. Add 2.0 ml NaOH solution or a volume sufficient to produce pH 12-13

3. Mix, add 2 drops murexide indicator and mix again

4. Titrate with EDTA slowly, with continuous stirring to the proper end-point. The colour will
change from red to violet. Check end-point by adding 1 to 2 drops of titrant in excess to
make certain that no further colour change occurs. Facilitate end-point recognition by
preparing a colour comparison blank containing 2.0 mL NaOH solution, 2 drops indicator
and sufficient EDTA titrant (0.05-0.10 mL) to produce an unchanging colour.

Remark:
Because of the high pH used in this procedure, titrate immediately after adding NaOH
and indicator.

3.3.5 Calculation
1000
mg Ca/L = (V x M x 40.08) x , where
mL sample
V = mL EDTA titrated
M = molarity of EDTA

Literature:
American Public Health Organisation, Standard Methods for the examiniation of water and
wastewater, 18th edition 1992 p.3-57/3-58.

26
Determination of cations

3.4 Ca + Mg (Hardness); EDTA Titrimetric Method

3.4.1 Principle

If Eriochrome Black T is added to a water sample containing Ca and Mg ions at a pH 10.0


± 0.1, the solution becomes wine red. If EDTA is added, the Ca and Mg will be complexed,
and when all of the Ca and Mg has been complexed the solution turns from wine red to blue,
marking the end-point of the titration.
Mg ion must be present to yield a satisfactory end-point. To insure this, a small amount of
complexometrically neutral Mg-EDTA is added to the buffer; this automatically introduces
sufficient Mg. A limit of 5 min. is set for the duration of the titration to minimize the tendency
toward CaCO3 precipitation.

3.4.2 Interference

Some metal ions interfere by causing fading or indistinct end-points or by consumption of


EDTA. This interference can be reduced by the addition of certain inhibitors before titration.
With heavy metal or polyphosphate concentrations below those indicated in Table 1, use
inhibitor I or II.
When higher concentrations of heavy metals are present, determine Ca and Mg with the
atomic absorption technique.

Table I
Maximum concentrations of interferences permissible with various inhibitors.
Values based on using 25 mL sample diluted to 50 mL

Max. Interference conc. mg/L


Interfering substance Inhibitor I Inhibitor II

Al 20 20
Cd 20
Co over 20 0.3
Cu over 30 20
Fe over 30 5
Pb 20
Mn2+ 1
Ni over 20 0.3
Zn 200
Polyphosphate 10

Suspended or colloidal organic matter also may interfere with the end-point. Eliminate this
interference by evaporating the sample to dryness on a steam bath and heating in an oven
at 550°C until the organic matter is completely oxidized. Normally 15-20 min. is long enough
to oxidize 200 mg of solids. Dissolve the residu in 20 mL 1 M HCl, neutralize to pH 7 with
1 M NaOH and make up to 50 mL with H2O. Cool to room temperature and continue
according to procedure 3.4.4.

3.4.3 Reagents

a) Buffer: 1. Dissolve 1.179 g Na2EDTA.2H2O and 780 mg MgSO4.7H2O in 50 mL.


2. Dissolve 16.9 g NH4Cl in 143 mL conc. NH4OH.
Add solution 1 to solution 2 with mixing and dilute to 250 mL.
Store this solution for no longer than 1 month in a plastic or borosilicate glass
bottle.

27
Determination of cations

Keep closed as much as possible to prevent loss of NH3 or uptake of CO2.


Discard buffer solution when 1 or 2 mL added to the sample fails to produce a
pH of 10.0 ± 0.1 at the titration end-point.

b) Complexing agents: Occasionally water containing interfering ions require adding an


appropiate complexing agent to give a clear, sharp change in colour
at the end-point. If necessary use inhibitor I or II.

1. Inhibitor I: Adjust acid samples to pH 6 or higher with 0.1 M NaOH or buffer. Add 250
mg NaCN (or KCN), to 25 mL sample diluted to 50 mL as described in 3.4.4, add
sufficient buffer to adjust pH 10.0 ± 0.1

1
CN (cyanide) is extremely poisonous, take extra precautions in its
use. Flush solutions containing this inhibitor with large quantities
of water after insuring that no acid is present in the collecting
container. Under acidic conditions HCN
(= hydrogen cyanide) will be liberated. This is a very poisonous
gas.

2. Inhibitor II: Dissolve 5.0 g Na2S.9H2O or 3.7 g Na2S.5H2O in 100 mL. Close glass
bottle with a rubber stopper, this inhibitor deteriorates through air oxidation. It
produces a sulfide precipitation that obscures the end-point when high concentrations
of heavy metals are present. Use 1 mL in procedure 3.4.4 before adding buffer.

c) Eriochrome black T: Dissolve 0.5 g in 90 mL triethanolamine.

d) EDTA 0.01 M: Dissolve 3.723 g Na2EDTA.2H2O in 1000 mL. Standardize this solution.
1 mmol EDTA = 1 mmol Ca. Store preferable in polyethylene bottle
because the EDTA extracts hardness producing cations from soft-glass
bottles. Restandardize from time to time.

3.4.4 Procedure

1. Take a sample volume that requires less than 15 mL EDTA and complete titration within
5 min, measured from the time of buffer addition.
Dilute 25.0 mL sample to about 50 mL in a borosilicate erlenmeyer
2. Add 1-2 mL buffer, usually 1 mL will be sufficient to produce a pH 10.0 ± 0.1
3. Add 2 drops eriochrome black T indicator
4. Titrate slowly with EDTA, with continuous stirring, until the last reddish tinge disappears.
Add the last few drops at 3-5 sec. intervals.

3.4.5 Calculation
1000
Hardness as mg CaCO3/L = (V x M x 100) x , where
mL sample

V = mL EDTA titrated
M = molarity of EDTA

28
Determination of cations

3.4.6 Remarks

1. Titrate at room temperature. The colour change becomes very slow at freezing
temperature and indicator decomposition occurs in hot water
2. The absence of a sharp end-point means that an inhibitor must be added or that the
indicator has deteriorated
3. Titrate under daylight conditions since ordinary incandescent lights tend to produce a
reddish tinge in the blue at the end-point.
4. For low hardness samples (less than 5 mg/L) take a larger sample volume i.e.
100-1000 mL. Add proportionally larger amounts of buffer, inhibitor and indicator. Titrate
in that case also a blank and compensate
5. If the approximate hardness is known, precipitation loss can be reduced by adding 90%
or more of EDTA to the sample before adjusting pH with buffer.

Literature:
American Public Health Organisation
Standard Methods for the examination of water and wastewater, 18th edition 1992
p. 2-36/2-38.

29
Determination of cations

3.5 Fe; phenanthroline method

3.5.1 Principle

Iron is brought into solution, reduced from Fe3+ to Fe2+ by boiling with acid and
hydroxylamine. Fe2+ will form an orange-red coloured complex with 1,10 phenanthroline.
The intensity of the colour is independent of pH 3 to 9. A pH between 2.9 and 3.5 insures
rapid colour development in the presence of an excess of phenanthroline.

3.5.2 Interference

Strong oxidizing agents, CN-, NO2-, phosphates (polyphosphates more than o-PO43-), Cr, Zn
in concentrations exceeding 10 times that of iron, Co and Cu in excess of 5 mg/L and Ni in
excess of 2 mg/L are interfering substances.
Bi, Cd, Hg, Ag and molybdate precipitate phenanthroline.
The initial boiling with acid converts polyphosphates to orthophosphate and removes CN-
and NO2- that otherwise would interfere. Adding excess hydroxylamine eliminates errors
caused by strong oxidizing agents.
In the presence of interfering metal ions, use more phenanthroline to compensate for the
amount which forms complexes with those ions. Where excessive concentrations of
interfering metal ions are present, the extraction method may be used. The presence of
excessive amounts of organic matter may require digestion before use of the extraction
procedure.

3.5.3 Detection limit

The detection limit is 10 μg/L with cells of 5 cm.

3.5.4 Apparatus

a) Spectrophotometer, for use at 510 nm with a 1 cm cell (or longer)


b) Acid-washed glassware: Wash all glassware with conc. HCl and rinse with distilled H2O
before use. This removes deposits of iron oxide
c) Separatory funnels, only when samples are digested

3.5.5 Reagents

Store reagents in glass bottles, the HCl and ammonium acetate are stable indefinitely if
tightly stoppered.
The hydroxylamine, phenanthroline and stock iron solutions are stable for several months.
The standard iron solutions are not stable; prepare daily as needed by diluting the stock
solution.

a) HCl, conc. Containing less than 0.00005% Fe (= 0.5 mg/L)


b) Hydroxylamine: Dissolve 10 g NH2OH.HCl in 100 mL
c) Ammoniumacetate buffer: Dissolve 250 g NH4C2H3O2 in 150 mL, add 700 mL conc.
acetic acid. Prepare a new calibration graph with each new buffer preparation
d) Phenanthroline: Dissolve 1.0 g 1,10 phenanthroline monohydrochloride monohydrate
(C12H9N2Cl.H2O) in 1 L; one mL of this complexing reagent is sufficient for no more than
100 μg Fe

30
Determination of cations

e) Stock Fe: Slowly add 20 mL conc. H2SO4 to 50 mL H2O and dissolve 1.404 g
(NH4)2Fe(SO4)2.6H2O. Add dropwise 0.1 M KMnO4 until a faint pink colour persists. Dilute
to 1000 mL with H2O and mix. 1.00 mL= 200 μg Fe

f) Standard Fe: Prepare daily for use; dilute 50.00 mL stock Fe to 1000 mL.
1.00 mL = 10 μg Fe
Reagents h and i are required when the samples are digested

g) Sodiumacetate: Dissolve 200 g NaC2H3O2.3H2O in 800 mL


h) Di-isopropyl ether.

3.5.6 Calibration

1. Add volumes in the range from 0-50.00 mL of standard Fe (1.00 mL = 10 μg Fe) to


100 mL erlenmeyer flasks and dilute to 50 mL
2. Add 2 mL conc. HCl, 1 mL NH2OH.HCl and some pumice grains
3. Heat to boiling until the volume is reduced to 15-20 mL
4. Cool to room temperature and transfer to 100 mL volumetric flasks
5. Add 10 mL ammonium acetate buffer and 20 mL phenanthroline
6. Dilute to the mark and mix, measure the absorbance against H2O within 5 to 10 min. in a
1 cm cell at 510 mn
7. Prepare a calibration line by plotting the absorbance against μg Fe/100 mL endvolume.
Determine the mathematical expression for this line.

3.5.7 Procedure for total Fe

1. Add 50.00 mL sample or a smaller accurately measured portion which does not contain
more than 0.5 mg Fe to a 125 mL erlenmeyer flask. Dilute, if necessary, to 50 mL.

Remark: If noticeable amounts of colour or organic matter are present, it may be necessary
to evaporate the sample in a porcelain dish, gently ash the sample during 15 min at 550°C,
and redissolve the ash in 2 mL conc. HCl and 5 mL H2O. Pretreat the porcelain dish by
boiling for several hours in 1 + 1 HCl.

2. Continue this procedure by starting with step 2 mentioned under calibration (3.5.6).

3.5.8 Procedure for total dissolved Fe

1. Immediately after collection filter sample through a 0.45 μm membrane filter into a
vacuum flask containing 1 mL conc. HCl/100 mL sample
2. Analyze this filtrate according to procedure 3.5.7.

31
Determination of cations

3.5.9 Procedure for Fe2+

Determine Fe2+ at the sampling site because of the possibility of change in the Fe3+/Fe2+
ratio with time in acid solutions.

1. Add 50.00 mL sample or a smaller accurately measured volume to a 100 mL volumetric


flask which already contains 1 mL conc HCl/50 mL sample
2. Add 10 mL ammonium acetate buffer and 20 mL phenanthroline with vigorous mixing
3. Dilute to 100 mL and measure the absorbance within 5 to 10 min.

Remark: do not expose orange-red complex to sunlight.

3.5.10 Remark

If samples are coloured or turbid, also carry out the whole procedure for a second set of
samples. However, do not add phenanthroline to this second set. Measure the absorbance
and translate this into iron values.
Subtract these values for the values found with the addition of phenanthroline. This
procedure does not compensate for interfering ions!

3.5.11 Treatment for samples containing organic interferences

1. Digest samples according to procedure 2.4.2.


2. Pipet 10.00 mL or other suitable portion containing 20-500 μg Fe from the 250 mL
volumetric flask to a separatory funnel. If the volume taken is less than 10 mL, add H2O
to make up to 10 mL
3. Add 1.5 mL conc. HCl/ mL sample
4. Add 25 mL di-isopropylether (caution) and shake for 30 sec.
5. Draw off lower acid layer into a second separatory funnel
6. Extract acid solution again with 25 mL di-isopropylether, drain acid layer into clean
beaker and combine the two portions of di-isopropylether
7. Pour acid layer back into second separatory funnel and extract again with 25 mL
di-isopropylether
8. Discard acid layer and add ether layer to the first separatory funnel
9. Shake combined ether extracts with 25 mL H2O to return Fe to aqueous phase and
transfer lower aqueous phase to 100 mL volumetric flask
10. Repeat step 9 and add aqueous layer to the same 100 mL volumetric flask
11. Discard the ether layer
12. Add 1 mL NH2OH.HCl, 10 mL Na-acetate! and 10 mL phenanthroline. Fill up to the mark
and mix
13. Measure the absorbance after 10 min. at 510 nm against H2O
14. Prepare a calibration line, let the standards follow the entire digestion and extraction
procedure!

Remark:
Carry out the extraction procedure in the fume cupboard.

32
Determination of cations

3.5.12 Calculations

When the sample has been treated according to 3.5.7; 3.5.8 and 3.5.9.

μg Fe ( in 100 mL final volume )


mg Fe/L =
mL sample

When the sample has been treated according to 3.4.11

250
mg Fe/L = μg Fe (in 100 mL final volume) x
sample portion (10 ) x sample volume ( 50 )

= μg Fe (in 100 mL final volume) x 0.5 when the sample portion is 10 mL

Fe3+ = total Fe - Fe2+

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 3-66/3-68.

33
Determination of cations

3.6 K; flame emission photometry

3.6.1 Storage of samples

Do not store samples in soft-glass bottles because of the possibility of contamination from
leaching of the glass. Use acid-washed polyethylene or borosilicate glass bottles. Adjust
sample to pH<2 with HNO3, this will dissolve potassium salts and reduce adsorption on
vessel walls.

3.6.2 Principle

Trace amounts of K can be determined in a direct reading type of flame photometer at a


wavelength of 766.5 nm and a slit width of 2 nm. Minimum detectable concentration is
approximately 0.1 mg/L.

3.6.3 Reagents, store in polyethylene bottles

Stock K: Dissolve 1.907 g KCl dried at 110° C and dilute to 1000 mL, 1 mL = 1.00 mg K
Standard K: Dilute 10.00 mL stock solution to 100 mL, 1 mL = 0.100 mg K

3.6.4 Standard serie, store in polyethylene bottles

Dilute 0-10.00 mL of standard K to 100 mL


This will cover the range of 0-10 mg/L

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 3-80.

34
Determination of cations

3.7 Na; flame emission photometry

3.7.1 Storage of samples

Do not store samples in soft-glass bottles because of the possibility of contamination from
leaching of the glass. Use acid-washed polyethylene or borosilicate glass bottles. Adjust
sample to pH<2 with HNO3, this will dissolve sodium salts and reduce adsorption on vessel
walls.

3.7.2 Principle

Trace amounts of Na can be determined in a direct reading type of flame photometer at a


wavelength of 589 nm and a slit width of 0.7 nm. Minimum detectable concentration is
approximately 0.1 mg/L.

3.7.3 Glassware

All acid rinsed with HNO3 and H2O.

3.7.4 Reagents, store in polyethylene bottles

Stock Na: Dissolve 2.542 g NaCl dried at 140°C and dilute to 1000 mL. 1 mL = 1.00 mg Na
Standard Na: Dilute 10.00 mL stock to 100 mL. 1 mL = 0.100 mg Na

3.7.5 Standard serie, in polyethylene bottles

Dilute 0 - 10.00 mL of standard Na to 100 mL.


This will cover the range of 0 - 10 mg/L.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 3-93/3-95.

35
Determination of anions

4. DETERMINATION OF ANIONS

4.1 HCO3- / CO32- ; Alkalinity; Titrimetric analysis

4.1.1 Background

The buffering capacity of water is an important characteristic for water quality.


The term alkalinity is also directly involved in water and wastewater unit operations such as
water softening, coagulation, iron removal and pH neutralization. In natural waters alkalinity
may generally be associated with the carbonate equilibrium; most important sources are
then the atmospheric CO2 and limestone, CaCO3.
At pH ≥ 12, CO32- is exclusively present; at pH =8.3 it is all HCO3- and at pH ≤ 4.5 it is all
CO2.
Alkalinity is defined as the amount of acid necessary to reach the above equivalence points.
Since the first equivalence point, at pH 8.3, often the colour indicator phenolphthalein is
used, the corresponding alkalinity is referred to as the ‘phenolphthalein alkalinity’, the
amount of acid necessary to reach pH 4.5 is then called ‘total alkalinity’.
Also metacresol purple can be used as indicator instead of phenolphthalein.
Methyl orange or the mixed indicator bromocresol green-methyl red will be used to detect
the second equivalence point at pH 4.5
For historical reasons alkalinity is often expressed as mg CaCO3 /L.

4.1.2 Sample treatment

Do not filter, dilute, concentrate, or alter the sample.

4.1.3 Reagents

a) Phenolphthalein indicator: Dissolve 0.5 g in 50 mL ethanol and 50 mL H2O.


b) Metacresol purple indicator: Dissolve 100 mg in 100 mL H2O.
c) Methyl orange indicator: Dissolve 50 mg in 100 mL H2O.
d) Bromocresol green- methyl red indicator: Dissolve 100 mg bromocresol green and 20 mg
methyl red in 100 mL ethanol.
e) 0.020 N HCl or 0.02 N H2SO4 ( = 0.01 M H2SO4 )

4.1.4 Procedure

1. Transfer, with a pipet, a known volume of sample to an erlenmeyer flask.


2. Add about 5 drops phenolphthalein indicator.
3. In case a red colour appears (pH ≥ 8.3) : titrate with 0.020 N HCl until the red colour
disappears and record the volume used.
Add to the same solution about 5 drops methyl orange or the mixed indicator and titrate
until a red colour appears, record the volume used.
4. In case no colour will appear ( pH ≤ 8.3): add about 5 drops methyl orange or the mixed
indicator and titrate until a red colour appears, record the volume used.

37
Determination of anions

4.1.5 Calculations

V * N * 1000
Alkalinity as mg/L CaCO3 = * 100
mL sample * 2

V * N * 1000
In case initial pH ≤ 8.3 : mg/L HCO3- = * 61
mL sample

V = titration volume in mL,


N = normality of the acid solution,
100 = molecular mass of CaCO3,
61 = molecular mass of HCO3- .

38
Determination of anions

4.2 Cl-; argentometric titration

4.2.1 Principle

In a neutral or slightly alkaline solution, potassiumchromate (K2CrO4) can indicate the


endpoint of the AgNO titration of Cl-.
3
AgCl is precipitated quantitatively before red Ag2CrO4 is formed.

4.2.2 Interference

Substances in amounts normally found in potable waters will not interfere. Br-, I- and CN-
register as equivalent Cl-. Sulfide, S O 2- and SO - interfere but can be removed by
2 3 3
treatment with H2O2. Ortho-PO4 in excess of 25 mg/L interferes by precipitating as Ag3PO4.
Fe in excess of 10 mg/L interferes by masking the endpoint. A potentiometric method is
suitable for coloured or turbid samples in which the colour-indicated endpoint might be
difficult to observe. However, a turbidity and colour can also be removed by treatment with
Al(OH)3.

4.2.3 Reagents

a) K2CrO4 indicator: Dissolve 12.5 g K2CrO4 in a little H2O, add AgNO3 solution
until a definite red precipitate is formed. Let stand 12 h, filter
and dilute to 250 mL.
b) AgNO3 titrant: Dissolve 2.40 g AgNO3 in water and dilute to 1000 mL. Store
in a brown bottle. Standardize against NaCl;
1 mol Ag+ = 1 mol Cl-
c) NaCl standard: Dissolve 824 mg NaCl, dried at 140° C, in water and dilute to
1000 mL. 1 mL = 0.0141 mmol Cl-.
d) Phenolphthaline indicator: Dissolve 0.5 g in 50 mL ethanol and add 50 mL H2O
e) NaOH 1 M: Dissolve 4 g in 100 ml H2O
f) H2SO4 0.5 M: Add 2.7 mL conc. H2SO4 to 100 mL H2O
g) H2O2 30%
h) Al(OH)3 suspension: Dissolve 30 g AlK(SO4)2.12H2O in 250 mL H2O. Heat to 60°C
and add 14 mL conc. NH4OH slowly with stirring. Let stand
about 1 h, transfer to a large bottle and wash precipitate by
successive additions, with thorough mixing and decanting with
water, until free from chloride. The suspension occupies a
volume of approximately 250 mL.

4.2.4 Standardization of AgNO3

1. Pipet 10.00 mL NaCl standard in an erlenmeyer flask of 250 mL


2. Add about 90 mL H2O, adjust to pH 7-10 if necessary
3. Add 1 mL K2CrO4 indicator
4. Titrate with AgNO3 to pinkish yellow endpoint. Be consistent in endpoint recognition

39
Determination of anions

0 .141
Molarity AgNO3 =
mL titrant

4.2.5 Procedure

1. Use a 100 mL sample or a suitable portion diluted to 100 mL which contains 0.15 - 10 mg
Cl-
2. If the sample is highly coloured or turbid, add 3 mL Al(OH)3 suspension, mix, let settle
and filter. If sulfide, sulfite or thiosulfate is present, add 1 mL H2O2 and stir for 1 min.
3. Adjust pH 7-10, if necessary
4. Add 1 mL K2CrO4 indicator
5. Titrate with AgNO3, be consistent in endpoint recognition
6. Carry out a blank, a titration volume for the blank of 0.2-0.3 mL is usual.

4.2.6 Calculation

1000
mg Cl-/L = (A - B) x M x 35.45 x , where
mL sample

A = mL titration for sample


B = mL titration for blank
M = molarity of AgNO3

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-49/4-50.

40
Determination of anions

4.3 NO3 ; Procedures

4.3.1 NO3-; Spectrophotomretric method with 2,6-dimethylphenol

4.3.1.1 Apparatus

a) Spectrophotometer for use at 324 nm with 1 cm cells

4.3.1.2 Reagents

a) Reagent 1: Dissolve 0.040 g amidosulfonic acid in 10 mL H2O


Transfer to a 2L beaker and add 500 mL conc. H2SO4 and
500 mL conc. H3PO4

b) Reagent 2: Dissolve 1.2 g 2,6-dimethylphenol in 1000 mL glacial


acetic acid
c) Stock NO3-N 1000 mg/L : Dissolve 7.218 g KNO3 (dried at 105ºC for 24 h) in H2O
and dilute to 1000 mL.
d) Standard NO3-N 100 mg/L: Dilute 10.00 mL stock solution to 100.0 mL with H2O

4.3.1.3 Calibration

1) Transfer the following amounts of standard NO3-N to 100 mL volumetric flasks: 0; 1.00;
5.00; 10.00; 15.00; 20.00; 25.00 and 30.00 mL and fill up to the mark.
The concentration range is 0 – 30 mg NO3-N/L.
2) Add 35 mL of Reagent 1 to a 50 mL volumetric flask
3) Add 5.00 mL of each solution from 1) to a 50 mL volumetric flask
4) Mix well and wait for 5-10 minutes
5) Add 5.00 mL of Reagent 2. Mix well and wait for 10 minutes
6) Measure the absorbance at 324 nm between 10 – 60 minutes with a 1 cm quartz cuvet
against H2O
7) Plot the absorbance against the NO3-N concentration (max. 30 mg/L) and determine the
mathematical expression of the calibration line.

4.3.1.4 Procedure for the sample

1. Filter the sample over a GF-52 filter


2. Follow the procedure as described in the Calibration, starting with step 2. Of course, in
step 3 one should add 5.00 mL filtered sample to the 50 mL volumetric flask
3. Use the calibration line to calculate the NO3-N concentration in the sample

41
Determination of anions

Remark:
1) In case the availability of the sample volume is limited it is possible to use reduced
volumes. Reduce the volumes of the solutions by a factor 5 and use 25 mL volumetric
flasks instead.
2) Follow the instructions with respect to colour development times carefully.

Standard deviation and recovery

A tapwater sample was analyzed 6 times.


The mean concentration found was 3.87 mg NO3-N /L with a relative standard deviation of
0.5%.
For a standard addition of 3.0 mg/L a recovery of 105% was found.

A surface water sample was analyzed 6 times.


The mean concentration found was 1.79 mg NO3-N /L with a relative standard deviation of
3%.
For a standard addition of 3.0 mg/L a recovery of 105% was found.

A wastewater effluent sample was analyzed 6 times.


The mean concentration found was 4.39 mg NO3-N /L with a relative standard deviation of
0.7%.
For a standard addition of 3.0 mg/L a recovery of 104% was found.

Literature:
Nitrate measurement according to ISO 7890/1 – 1986 (E)

42
Determination of anions

4.3.2 NO3-; Cadmium reduction spectrophotometric method

4.3.2.1 Principle

NO3- is reduced almost quantitatively to NO2- in the presence of Cd. The NO2- produced
thus is determined by diazotizing with sulfanilamide and coupling with N-(1-naphthyl)-
ethylene diamine dihydrochloride to form a highly coloured azo-dye that is measured
colorimetrically.
A correction may be made for any NO - present in the sample by analyzing without the
2
reduction step.
The range of this method is 0.01 - 1.0 mg NO3- - N/L.

4.3.2.2 Interference

Suspended matter in the reduction column will restrict sample flow. Concentrations of Fe,
Cu, or other metals above several mg/L lower reduction efficiency. Add EDTA to samples to
eliminate this interference. Oil and grease will coat the Cd surface, remove them by pre-
extraction with an organic solvent.
Residual chlorine can interfere by oxidizing the Cd column, reducing its efficiency. Sample
colour that absorbs at about 540 nm interferes.

4.3.2.3 Storage of samples

If storage is necessary, store for up to 24 h at 4°C. For longer storage, preserve with 2 mL
conc. H2SO4/L and store at 4°C.
When sample is preserved with acid, NO - and NO - cannot be determined as
3 2
individual species.

4.3.2.4 Apparatus

a) Reduction column
b) Spectrophotometer for use at 543 nm, cells of 1 cm

4.3.2.5 Reagents

a) Cu-Cd granules: Wash 25 g new or used 40-60 mesh (0.25-0.5 mm) Cd granules
with 6 M HCl and rinse with water. Add 100 mL 2% CuSO4 solution
and swirl for 5 min. or until blue colour partially fades. Decant and
repeat with fresh CuSO4 until a brown colloidal precipitate begins
to develop. Gently flush with water to remove all precipitated Cu.
b) Colour reagent: To 200 mL H2O add 25 mL 85% H3PO4 and 2.5 g sulfanilamide.
After dissolving completely add 0.25 g N-(1-naphthyl)-
ethylenediamine dihydrochloride. Mix to dissolve, then dilute to 250
mL. This solution is stable for about one month when stored in a
dark bottle in the refrigerator.
c) NH4Cl-EDTA: Dissolve 13 g NH4Cl and 1.7 g Na2 EDTA in 900 mL, adjust to pH
8.5 with conc. NH4OH and dilute to 1 L
d) Dilute NH4Cl-EDTA: dilute 300 mL NH4Cl-EDTA to 500 mL
e) CuSO4 2%: Dissolve 10 g CuSO4.5H2O in 250 mL H2O and dilute to 500 mL

43
Determination of anions

f) Stock NO3-: Dissolve 0.722 g KNO3 (dried at 105°C for 24 h) in water and dilute
to 1000 mL. Add 2 mL CHCl . 1 mL = 100 μg NO --N. This solution
3 3
is stable for at least 6 months

g) Standard NO3-: Dilute 100 mL stock to 1000 mL. Add 2 mL CHCl3. 1 mL = 10 μg


NO --N. This solution is stable for 6 months
3

h) Stock NO2-: Dissolve 1.232 g NaNO2 in water and dilute to 1000 mL. Add 1 mL
CHCl . 1 mL = 250 μg NO --N. Because NO - is oxidized readily in
3 2 2
the presence of moisture, use a fresh bottle of reagent for
preparing the stock solution and keep bottles tightly stoppered
against the free access of air when not in use. However, in order to
know the exact NO - content of this solution, this stock solution
2
must be standardized according to procedure 4.2.6 before adding
CHCl3

i) Standard NO2-: Dilute 2.00 mL stock NO2- to 1000 mL. Depending on the exact
NO - content in the stock the NO --N content will be about 0.5 μg
2 2

NO2- N/mL.

4.3.2.6 Standardization procedure for stock NO2-

Reagents

0.02 M KMnO4, see appendix 4.


2 M H2SO4, add 110 mL conc. H2SO4 to 900 mL H2O

1) Pipet 5.00 mL 0.02 M KMnO4 in a 300 mL erlenmeyer, add 50 mL 2 M H2SO4 and


100 mL H2O
2) Heat to 40-45ºC and titrate with the stock NO - - N solution.
2
Titrate the nitrite very slowly with constant stirring toward the end of the titration when the
solution has a pale violet colour. Titrate to colourless.

5 .00 x M M
NO2- - N μg/mL = x 14 x 1000 x 2.5 = x 175,000
Vt Vt

M = molarity KMnO4
Vt = titrated volume of stock nitrite solution

4.3.2.7 Pretreatment of reduction column

1) Add H2O to the column


2) Add sufficient Cu-Cd granules to produce a column of 18.5 cm long
3) Maintain water level above Cu-Cd granules to prevent entrapment of air
4) Wash column with 200 mL dilute NH4Cl-EDTA

5) Activate column by passing through it, at 7-10 mL/min, at least 100 mL of a solution
composed of 25% 1.0 mg NO3--N/L standard and 75% NH4Cl-EDTA.

44
Determination of anions

Remark:
There is no need to wash columns between samples, but if columns are not to be reused for
several hours or longer, pour 50 mL dilute NH4Cl-EDTA solution on the top and let it pass
through the system. Store Cu-Cd column in this solution and never let it dry.

4.3.2.8 Calibration procedure for NO3- reduction and measurement

1) Prepare a standard serie of 0; 2.5; 5.0; 10.0; 25.0 and 50.0 μg NO3--N in 50 mL by
diluting 0; 0.25; 0.50; 1.00; 2.50 and 5.00 mL standard NO3--N to 50 mL in volumetric
flasks
2) To 25.00 mL standard add 75 mL NH4Cl-EDTA and mix
3) Pour into the column and collect effluent at a rate of 7-10 mL/min
4) Discard the first 25 mL
5) Collect the rest in a flask
6) To 50.00 mL effluent add 2.0 mL colour reagent and mix. Do this as soon as possible
after the reduction procedure, wait not more than 15 min.
7) Measure the absorbance, between 10 min and 2 h, at 543 nm against H2O in a 1 cm cell.
Plot the absorbance against μg NO3--N in 50 mL (from step 1)
8) Determine the mathematical expression for this line
9) Compare at least one NO2- standard to a reduced NO3- standard at the same
concentration to verify reduction column efficiency. Reactivate Cu-Cd granules with
CuSO4 acc. to 4.2.5 when reduction efficiency falls below 75%

4.3.2.9 Sample treatment procedure

1) Remove turbidity and adjust pH 7-9


2) To 25.00 mL sample or a portion diluted to 25.0 mL add 75 mL NH4Cl-EDTA and mix. If a
25
portion of a mL is used, the dilution factor will be . The next steps are the same as
a
mentioned under 4.2.8 step 3 and further.

Remark:
If NO --N concentration exceeds 1 mg/L, use remainder of reduced sample to make an
3
appropriate dilution and analyze again.

μg NO - - N in 50 mL ( read from graph ) 25


NO3--N mg/L =
3
x
50 a

4.3.2.10 Report

Report as miligrams oxidized N per liter (the sum of NO3--N + NO2--N) unless the
concentration of NO --N is separately determined and subtracted.
2

45
Determination of anions

4.3.3 NO3-; U.V. spectrophotometric screening method

4.3.3.1 Principle

Use this technique only for screening samples that have low organic matter contents; i.e.
uncontaminated natural waters and potable water supplies. Measurement of U.V. absorption
at 220 nm enables rapid determination of NO -. Because disolved organic matter also may
3

absorb at 220 nm and NO3- does not absorb at 275 nm, a second measurement made at
275 nm may be used to correct the NO - value. The extend of this emperical correction is
3
related to the nature and concentration of organic matter and may vary from one water to
another. Acidification with 1 M HCl prevents interference from hydroxide or carbonate
concentrations up to 1000 mg CaCO3/L.

4.3.3.2 Interference

Dissolved organic matter, surfactants, NO2-, Cr6+, chlorite and chlorate interfere. Chloride
has no effect on the determination.

4.3.3.3 Apparatus

Spectrophotometer for use at 220 and 275 nm with quartz cells of 1 cm.

4.3.3.4 Reagents

a) Standard NO3-: 1.00 mL = 10.0 μg NO3--N. See 4.2.5


b) HCl 1 M

4.3.3.5 Calibration

1) Dilute 0; 5.00; 10.00; 15.00; 20.00 and 25.00 mL standard NO3 to 50 mL in volumetric
flasks. Add 1 mL 1 M HCl and mix. See remark 2
2) Measure the absorbance at 220 nm and 275 nm against H2O. "Zero" the instrument for
both wavelengths (this “zero” setting depends of the type of instrument)
Remark: clean the quartz cells carefully with special tissues and use the same cell,
as much as possible, for measurement of the standards and samples
3) Subtract two times the absorbance reading at 275 nm from the reading at 220 nm to
obtain absorbance due to NO3-
4) Plot the absorbance against μg NO --N/50 mL and determine the mathematical
3
expression of this line.

46
Determination of anions

4.3.3.6 Procedure

1. To 50 mL clear sample, filter if necessary, or a portion diluted to 50 mL, add 1 mL 1 M


HCl and mix
2. Proceed as mentioned under Calibration

μg NO3 - N in 50 mL endvolume
mg NO3--N/L =
mL sample

Remark:
1. If correction value is more than 10% of the reading at 220 nm, do not use this method
2. The calibration graph is lineair up to 500 μg NO3 – N / 50 mL (= 10 mg NO3 – N / L)

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-85/4-91

Vogel’s Textbook of Quantitative Chemical Analysis, p. 373-374


ISBN 0-582-44693-7

Quantitative Chemical Analysis, fourth edition by Kolthoff, Sandel, Meehan, Bruckenstein,


p. 825 and 834

47
Determination of anions

4.4 NO2-, spectrophotometric method

4.4.1 Principle

Nitrite (NO2-) is determined through formation of a reddish purple azo dye produced at pH
2.0-2.5 by coupling diazotized sulfanilamide with N-(1-naphthyl) ethylenediamine
dihydrochloride (NED dihychdrochloride)

4.4.2 Interferences

Chemical incompatibility makes it unlikely that NO2-, Cl2 and NCl3 will coexist. NCl3 imparts a
false red colour when colour reagent is added. The following ions interfere because of
precipitation under test conditions and should be absent: Fe3+, Pb2+, Hg2+, Ag+,PtCl62-
(chloroplatinate) VO32- (metavanadate), Sb3+, Au3+, Bi3+.
Cupric ion may cause low results by catalyzing decomposition of the diazonium salt.
Coloured ions that alter the colour system should be absent.
Remove suspended solids by filtration.

4.4.3 Storage of samples

Never use acid preservation for samples to be analyzed for NO2-. Make the determination
promptly on fresh samples to prevent bacterial conversion of NO - to NO - or NH .
2 3 3
For preservation for 1-2 days, freeze at -20°C or store at 4°C.

4.4.4 Apparatus

Spectrophotometer for use at 543 nm with 1 cm cells.

4.4.5 Reagents

a) Colour reagent: To 200 mL H2O add 25 mL 85% H3PO4 and 2.5 g sulfanilamide.
After dissolving completely add 0.25 g N-(1-naphthyl)-
ethylenediamine dihydrochloride. Mix to dissolve, then dilute to 250
mL. This solution is stable for about one month when stored in a
dark bottle in the refrigerator

b) Stock NO2-: Dissolve 1.232 g NaNO2 in water and dilute to 1000 mL. Add 1 mL
CHCl . 1 mL = 250 μg NO --N. Because NO - is oxidized readily in
3 2 2
the presence of moisture, use a fresh bottle of reagent for
preparing the stock solution and keep bottles tightly stoppered
against the free access of air when not in use. However, in order to
know the exact NO - content of this solution, this stock solution
2
must be standardized according to procedure 4.4.6 before adding
CHCl3

c) Standard NO2-: Dilute 2.00 mL stock NO2- to 1000 mL. Depending on the exact
NO - content in the stock the NO --N content will be about 0.5 μg
2 2

NO2- N/mL.

48
Determination of anions

4.4.6 Standardization procedure for stock NO2-

Reagents

0.02 M KMnO4, see appendix 4.


2 M H2SO4, add 110 mL conc. H2SO4 to 900 mL H2O

1) Pipet 5.00 mL 0.02 M KMnO4 in a 300 mL erlenmeyer, add 50 mL 2 M H2SO4 and


100 mL H2O
2) Heat to 40-45ºC and titrate with the stock NO - - N solution.
2
Titrate the nitrite very slowly with constant stirring toward the end of the titration when the
solution has a pale violet colour. Titrate to colourless.

5 .00 x M M
NO2- - N μg/mL = x 14 x 1000 x 2.5 = x 175,000
Vt Vt

M = molarity KMnO4
Vt = titrated volume of stock nitrite solution

4.4.7 Calibration

1) Dilute 0; 5.00; 10.00; 15.00; 20.00 and 25.00 mL standard NO2--N to 50 mL in volumetric
flasks. This serie is equal to 0.0; 2.5; 5.0; 7.5; 10.0 and 12.5 μg NO2--N/50 mL
2) Add 2 mL colour reagent and mix
3) Measure the absorbance between 10 min and 2 h at 543 nm against H2O in a 1 cm cell

4) Plot the absorbance against μg NO2--N/50 mL and determine the mathematical


expression of this line.

4.4.8 Procedure

1. Remove turbidity
2. If necessary, adjust pH to 5-9 with 1 M HCl or 1 M NH4OH
3. To 50.0 mL sample or a portion diluted to 50.0 mL add 2 mL colour reagent and mix
4. Measure the absorbance between 10 min and 2 h at 543 nm against water

μg NO2 - N in 50 mL endvolume
NO2--N mg/L =
sample volume

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-85/4-91

Vogel’s Textbook of Quantitative Chemical Analysis, p. 373-374


ISBN 0-582-44693-7

Quantitative Chemical Analysis, fourth edition by Kolthoff, Sandel, Meehan, Bruckenstein,


p. 825 and 834

49
Determination of anions

4.5 PO43-; ascorbic acid spectrophotometric method

4.5.1 Principle

Ammoniummolybdate and potassium antimonyl tartrate react in acid medium with


orthophosphate to form a phosphomolybdic acid that is reduced to intensely coloured
molybdenum blue by ascorbic acid. The minimum detectable concentration is 10 μg P/L with
use of a 5 cm cell. Phosphates that respond to colorimetric test without any treatment like
hydrolysis or oxidation (except filtration) are also called dissolved reactive phosphorus
(DRP). This reactive P is largely a measure of orthophosphate.

4.5.2 Interference

Arsenates react with the molybdate reagent to produce a blue colour similar to that formed
with phosphate.
Concentrations as low as 0.1 mg As/L interfere with the phosphate determination. Cr6+ and
NO - interfere to give results about 3% low at concentration of 1 mg/L and 10-15% low at
2
10 mg/L.
Na2S and silicate do not interfere at concentrations of 1 and 10 mg/L.

4.5.3 Sampling and storage

If phosphorus forms are to be differentiated, filter sample immediately after collection.


Preserve by freezing at or below -10ºC. Add 40 mg HgCl2/L to the samples, especially when
they are to be stored for long periods (HgCl2 is a hazardous substance; take appropriate
precautions in disposal).
Do not add either acid or CHCl3 as a preservative when phosphorus forms are to be
determined. If total P alone is to be determined, add 1 mL conc. HCl/L or freeze without any
additions.

4.5.4 Apparatus

a) Spectrophotometer for use at 880 nm with 1 cm cells


b) Acid-washed glassware: Phosphate contamination is common because of its absorption
on glass surfaces. Avoid using commercial detergents containing phosphate. Clean all
glassware with hot 1+5 HCl and rinse well with water.
Preferably, reserve the glassware only for phosphate determination, and after use, wash
and keep filled with water until needed.
If this is done, acid treatment is required only occasionally.

4.5.5 Reagents

a) H2SO4 2.5 M: Add 70 mL conc. H2SO4 to 420 mL H2O

b) Potassium antimonyl tartrate: Dissolve 1.3715 g K(SbO)C4H4O6.½ H2O in 400 mL H2O


and dilute to 500 mL in a volumetric flask. Store in a glass
stoppered bottle
c) Ammonium heptamolybdate: Dissolve 20 g (NH4)6Mo7O24.4H2O in 500 ml H2O. Store in
a glass stoppered bottle
d) Ascorbic acid 0.1 M: Dissolve 1.76 g ascorbic acid in 100 mL H2O. This solution
is stable for about 1 week at 4°C

50
Determination of anions

e) Combined reagent: Mix the above reagents in the following order:


125 mL 2.5 M H2SO4 + 12.5 mL K(SbO)C4H4O6.½ H2O +
37.5 mL (NH4)6 Mo7O24.4H2O + 75 mL ascorbic acid.
Mix after addition of each reagent and let reagents reach
room temperature before they are mixed. If turbidity forms
in the combined reagent, shake and let stand for a few
minutes until turbidity disappears before the next solution
is added. This reagent is only stable for 4 hours!
f) Stock P solution: Dissolve 439.0 mg KH2PO4 (dried at 105°C for a few
hours) in 1000 mL. 1 mL = 100 μg PO43--P
g) Standard P solution: Dilute 5.00 mL stock P to 100 mL. 1 mL = 5 μg P
h) Phenolpthaleine indicator: Dissolve 0.5 g in 50 mL ethanol and add 50 mL H2O

4.5.6 Calibration

1) Transfer the following amounts of standard P to 100 mL volumetric flasks: 0; 2.00; 3.00;
5.00; 10.00; 15.00 and 20.00 mL
2) Add 16 mL combined reagent, fill up to the mark and mix
3) Measure the absorbance between 10 - 30 min at 880 nm with a 1 cm cell against H2O

4) Plot the absorbance against μg P/100 mL and determine the mathematical expression of
the calibration line.

4.5.7 Procedure for o-PO43-; 0.1-2 mg P/L

1. Pipet 50.00 mL 0.45 μm filtered sample or less to a 100 mL volumetric flask, add 1 drop
phenolphthaleine indicator
2. If a red colour develops (pH >8.3) add dropwise 2.5 M H2SO4 to just discharge the
colour
3. Add 16 mL combined reagent, fill up to the mark and mix
4. Measure the absorbance at 880 nm between 10-30 min with a 1 cm cell.

Remark:
Natural colour of water generally does not interfere at the high wavelength used. For highly
coloured or turbid waters, prepare a blank by adding 8 mL 2.5 M H2SO4 and 2.4 mL
ammonium molybdate to 50.00 mL sample and fill up to 100 mL. Subtract blank absorbance
from sample absorbance.

μg P in 100 mL endvolume
mg P/L =
mL sample

4.5.8 Procedure for low o-PO43- content; 1-100 μg P/L

4.5.8.1 Additional Reagents

a) Standard P: Dilute 5.00 mL stock solution to 1000 mL. 1 mL = 0.5 μg P.


b) n-hexanol
c) isopropanol

51
Determination of anions

4.5.8.2 Calibration

1) Place into 250 mL separation funnels volumes of 0; 1.00; 2.00; 5.00; 10.00; 20.00;
30.00 and 40.00 mL standard P. (0 - 20 μg P)
2) Add H2O until endvolume is 200 mL
3) Add 20 mL mixed reagent, mix well and allow to stand for 10 minutes
4) Add 15.0 mL n-hexanol and shake the mixture vigorously for 15 min. in a mechanical
shaker
5) Reject the aqueous layer
6) Add 1.0 mL isopropanol, swirl the funnel gently to clear any water mist and reject any
further aqueous layer
7) Run off the solvent layer into a centrifuge tube and centrifuge for about 1 min.
8) Measure the absorbance at 680 nm! in 1 cm cells against n-hexanol
9) Plot the absorbance against μg P
10) Determine the mathematical expression of the calibration line.

4.5.8.3 Procedure

1. Place into a 250 mL separation funnel a quantity of 0.45 μm filtered sample not
exceeding 200 mL and containing between 0.25 and 20 μg P
2. Also carry out a blank
3. Follow steps 2-8 of the calibration procedure.

( A − B ) x 1000
μg P/L =
mL sample

where
A = μg P in sample
B = μg P in blank

Standard deviation and recovery procedure 4.4.8

A seawater sample (made of 3.3% seasalt in tapwater) was analyzed 5 times. The mean
concentration found was 16.9 μg P/L with a relative standard deviation of 2.7%.
For a standard addition of 5.0 μg P/L a recovery of 80% was found.

A brackish water sample was analyzed, the concentration was found to be 26.4 μg P/L.
For a standard addition of 30.0 μg P/L a recovery of 91% was found.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-108, 4-115/4-116.
Exam. of Water of Pollution control, Volume 2. Editor Michael J Suess, IHE book number
B3-79 p 310. ISBN 0-08-025255-9

52
Determination of anions

4.6 SO42-; turbidimetric method

4.6.1 Principle

SO42- is precipitated in an acetic acid medium with BaCl2 so as to form BaSO4 crystals of
uniform size.
Light absorbance or scattered light is measured by a photometer.
Minimum detectable concentration is approximately 1 mg/L.

4.6.2 Storage of samples

In the presence of organic matter certain bacteria may reduce SO42- to S2-. To avoid this,
store heavily polluted or contaminated samples at 4°C.

4.6.3 Interference

Colour or suspended matter in large amounts will interfere. Some suspended matter may be
removed by filtration. Silica in excess of 500 mg/L will interfere, and in waters containing
large quantities of organic material it may not be possible to precipitate BaSO4 satisfactorily.
In potable waters no other ions than SO42- will precipitate under strongly acid conditions.

4.6.4 Apparatus

a) Magnetic stirrer with constant stirring speed, use magnets of identical shape and size.
The exact speed is not critical, but keep it constant for standards and samples. Adjust
the speed to prevent splashing
b) Turbidity meter or spectrophotometer for use at 420 nm with 5 cm cells
c) Stopwatch
d) Measuring spoon, capacity 0.2 - 0.3 mL

4.6.5 Reagents

a) Buffer A: Dissolve 30 g MgCl2.6H2O, 5 g CH3COONa.3H2O, 1.0 g KNO3 and 20


mL CH3COOH in 500 mL H2O and make up to 1000 mL. Use this buffer
for samples containing > 10 mg SO42-/L.
b) BaCl2 crystals of 20-30 mesh (0.6 - 0.8 mm).
In standardization, uniform turbidity is produced with this mesh size range and the
appropriate buffer.
c) Stock SO42- : Dissolve 1.479 g dried Na2SO4 in 1000 mL. 1 mL = 1 mg SO42-.

d) Standard SO42- : Dilute 100 mL stock to 1000 mL 1.00 mL = 100 μg SO42-

Alternative : Dilute 10.4 mL 0.0200 N H2SO4 to 100 mL. 1.00 mL = 100 μg SO42-

53
Determination of anions

4.6.6 Calibration

1) Pipet 0;10.00; 15.00; 20.00; 25.00; 30.00; 35.00 and 40.00 mL standard in a 100 mL
volumetric flask and fill up to the mark. This covers the range of 0-4000 μg
SO42- / 100 mL.
2) Pour content of volumetric flask in a 250 mL beaker + magnet, add 20 mL buffer and
mix
3) While stirring, add a spoonful of BaCl2 and begin timing immediately

4) Stir for 60 ± 2 sec. at constant speed


5) After stirring period has ended, pour solution into the absorption cell and measure
turbidity at 5 ± 0.5 min
6) Plot turbidity against μg SO42-/100 mL.

4.6.7 Procedure for SO42- concentration between 10-40 mg/L

1. Measure 100 mL sample, or a suitable portion diluted to 100 mL in a volumetric flask,


into a 250 mL beaker + magnet, add 20 mL buffer and mix
2. Proceed as mentioned in the calibration procedure from step 3 on. Correct for sample
colour and turbidity by running blanks to which BaCl2 is not added. Translate turbidity to
equivalent SO42- concentration and subtract from sample result.

μg SO4 in 100 mL endvolume


mg SO42-/L =
mL sample

4.6.8 Procedure for SO42- concentration between 0-10 mg/L

Instead of buffer A another buffer, buffer B must be used.


Buffer B: Add to buffer A 0.111 g dried Na2SO4. Prepare a calibration curve by diluting 0;
1.00; 2.00; 3.00; 5.00; 7.00 and 10.00 mL standard to 100 mL in volumetric flasks.
This covers the range of 0-1000 μg SO42-/100 mL.
Proceed as mentioned under 4.6.6 and 4.6.7.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-134.

54
Oxygen

5. OXYGEN

5.1 Dissolved Oxygen (DO); Azide modification

5.1.1 General

The iodometric test is the most precise and reliable titrimetric procedure for DO analysis. It is
based on the addition of divalent manganese solution, followed by strong alkali, to the
sample in a glass-stoppered bottle. DO rapidly oxidizes an equivalent amount of the
dispersed divalent manganous hydroxide precipitate to hydroxides of higher valency states.
In the presence of iodide ions in an acidic solution, the oxidized manganese reverts to the
divalent state, with the liberation of iodine equivalent to the original DO content. The iodine is
then titrated with a standard solution of thiosulfate.
Use the azide modification for most wastewater, effluent and stream samples, especially if
samples contain more than 50 μg NO - -N/L and not more than 1 mg ferrous iron/L. Other
2

reducing or oxidizing materials should be absent. If 1 mL NaF solution (= 80 mg F-) is


added before the sample is acidified and there is no delay in titration, ferric iron in the range
of 100-200 mg/L will be complexed in such a way that it will not give any errors.

5.1.2 Reagents

a) MnSO4: Dissolve 36.4 g MnSO4.H2O in water, filter, and dilute to 100


mL. This solution should not give a colour with starch when
added to an acidified KI solution
b) Alkali-iodide-azide reagent: Dissolve 50 g NaOH and 15 g KI in water and dilute to 100
mL. Add 1 g NaN3 dissolved in 4 mL water. This reagent
should not give a colour with starch when diluted and
acidified
c) H2SO4 conc: 1 mL is equivalent to about 3 mL alkali-iodide-azide reagent
d) Starch 2%: Dissolve 2 g starch in 100 mL hot (boil!) water. Add 0.2 g
salicylic acid
e) Na2S2O3 0.025 M: Dissolve 6.205 g Na2S2O3.5 H2O in water, add 0.4 g NaOH
and dilute to 1000 mL. Standardize this solution.
1 mL 0.025 M Na2S2O3 ≈ 0.2 mg DO
f) NaF: Dissolve 18 g NaF in 100 mL water.

5.1.3 Procedure

1. Collect the sample carefully in a 250-300 mL bottle with known volume


2. Add 1.0 mL MnSO4 followed by 1.0 mL alkali-iodide-azide, hold pipet tips just above the
liquid
3. Stopper carefully to exclude air bubbles and mix by inverting the bottle a few times
4. Let precipitate settle (about half bottle volume) and discard supernatant
5. If necessary, add 1 mL NaF, and swirl gently
6. Add 1.0 mL conc. H2SO4 and swirl gently to dissolve the flocs
7. Titrate with 0.025 M Na2S2O3 to pale straw colour, add a few drops of starch and
continue titration to first disappearance of blue colour. Disregard subsequent

55
Oxygen

recolourations due to the catalytic effect of nitrite or to traces of ferric salts that have not
been complexed with fluoride.

5.1.4 Calculation

1000
DO mg/L = V x M x 8 x ; where
Vs − 2

V = mL Na2S2O3 used
M = molarity Na2S2O3
Vs =sample volume (= bottle volume)

5.1.5 Standardization of 0.025 M Na2S2O3 with K2Cr2O7

1. Add to an erlenmeyer flask 15 mL 10% KI solution and 20 mL 4 M HCl


2. Add 20.00 mL of a 1.2259 g/L K2Cr2O7 solution (Dried at 105ºC before weighing).
This K2Cr2O7 solution is equal to a 10 times diluted 0.0417 M solution which is
also used for COD determinations.
3. Add some NaHCO3 to remove oxygen and wait for 3 min.
4. Add 250 mL H2O and titrate, at the end of the titration 2 mL 0.5% starch solution must be
added.

Calculation

0 .500
Molarity Na2S2O3 =
mL Na2 S2 O3

The chemical reactions occurring during the Winkler titration are as follows:
I 2 Mn2+ + 2 H2O + O2 2 MnO2 ↓ + 4 H+
+ -
II 2 MnO2 + 8 H + 4 I 2 Mn2+ + 4 H2O + 2 I2
2-
III 4 S2O3 + 2 I2 2 S4O62- + 4 I-

Reaction II only occurs under acidic conditions.


So, one mole O2 is equivalent to 4 moles of S2O32-;
or 1 mmole S2O32- is equivalent to 0.25 mmole O2;
or 1 mmole S2O32- is equivalent to 0.25 x 32 mg O2.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 4-98/4-102.

56
Oxygen

5.2 Biochemical Oxygen Demand (BOD)

5.2.1 Preservation

If possible, analyze samples immediately. Otherwise, store at a low temperature (4°C)


immediately after collection to preserve most sample. Never use preservatives for samples
to be analyzed for BOD.

5.2.2 General Discussion

The biochemical oxygen demand (BOD) determination is an empirical test in which


standardized laboratory procedures are used to determine the relative oxygen requirements
of wastewaters, effluents, and polluted waters. The test measures the oxygen utilized during
a specified incubation period for the biochemical degradation of organic material
(carbonaceous demand) and the oxygen used to oxidize inorganic material such as sulfides
and ferrous iron. It also may measure the oxygen used to oxidize reduced forms of nitrogen
(nitrogenous demand) unless their oxidation is prevented by an inhibitor. The seeding and
dilution procedures provide an estimate of the BOD at pH 6.5 to 7.5. Determination of the
BOD520 of wastewater
The BOD of a given sample is the amount of O2, expressed in mg, consumed by micro-
organisms in 1 litre of a sample, when incubated in the dark at a fixed temperature for a fixed
period of time. Since the values obtained differ with temperature and period of incubation, these
have to be specified whenever a BOD value is given. In countries with moderate climates 5 days
incubation at 20°C is usual. BOD520 represents 60 - 65% of the total BOD.
Essentially, therefore, the BOD represents the amount of biodegradable organic matter
expressed in terms of the amount of oxygen required for its biological oxidation. The BOD is of
great practical importance as a basis for design of treatment plants, for taxation of communities
and industries discharging wastes and for assessing the impact of a given waste on the O2
balance of the receiving water.

From a theoretical point of view it is a most unsatisfactory characteristic, for which at present,
however, no better alternative is available. Theoretically, it is easy to calculate the amount of
oxygen necessary for complete biological oxidation of a known amount of a known organic
compound and this would include also the O2 consumed in mineralisation of the microbial cells
grown during the process. An experimental determination of this value would last a long time,
since the theoretical value is reached asymptotically.

In practice the composition of organic wastes is not known in qualitative and quantitative detail,
hence calculation is excluded. Also, for obvious reasons, the test cannot be extended to a period
of weeks. Hence a compromise must be made by shortening the incubation time to a practical
value and by assuming that in this period always the same percentage of the theoretical O2
consumption has taken place. This assumption is certainly not justified in many cases, as will be
clear from the following discussion of the factors affecting the result of a BOD determination.

Microbial population. Qualitatively this must consist of microbes capable of attacking the organic
matter present. If not available, such a population must be obtained in a preceding enrichment
experiment which then furnishes suitable ("acclimated") inoculation ("seed") material.
Quantitatively, the population must be large enough to overcome retardation in O2 consumption:
below a certain limit the size of the inoculum has a great influence on the time-course of growth
and O2 consumption. It should also be realized that in a latter phase of the test period O2 is
consumed in the mineralisation of bacterial cells by protozoa.

57
Oxygen

Composition of organic and inorganic matter present. Different organic compounds are not
mineralised at the same rate. Cellulose particles, for instance, are degraded much more slowly
than dissolved glucose. Some organic compounds inhibit the degradation of others, causing
sequential oxidation. Some intermediates or end products affect the pH or are inhibitory.
Inorganic substances like S2-, Fe2+, NH3 and NO2- are also liable to oxidation but may even
further complicate the picture as Fe2+ and S2- are also purely chemically oxidized and the
oxidation of NH3 and NO2- requires a very specific microbial population and usually proceeds at
a low rate. Obviously, absence of toxic components in the sample is necessary for a successful
test. Finally a reasonable balance of C, P and N sources must prevail during the entire test
period.

The BOD and the Chemical Oxygen Demand (COD) are entirely different characteristics since
the oxidation procedures applied in these two methods do not oxidize one and the same organic
compound to the same extent under the imposed test conditions and reaction period. Only in
cases where the composition of a given wastewater remains constant can an empirical ratio
COD/BOD be worked out that is practically useful. This ratio will also be influenced by the
presence of compounds toxic to microbes, which may lead to very low or zero values for the
BOD in cases where a high COD is found. In table 1 an overview is shown of COD and BOD of
some wastewaters.

Table 1. COD, BOD5, and BOD5/COD ratios of selected wastewaters


COD BOD BOD/COD
(mg/L) (mg/L)
Domestic sewage
Raw 500 300 0.60
After biological treatment 50 10 0.20
Slaughterhouse wastewater 3500 2000 0.57
Distillery vinasse 60000 30000 0.50
Dairy wastewater 1800 900 0.50
Rubber factory 5000 3300 0.66
Tannery wastewater 13000 1300 0.10
Textile-dying
Raw 1800 900 0.50
After biological treatment 120 5 0.04
Draft mill effluent
Raw 620 225 0.36
After biological treatment 250 30 0.12

Experimental procedure
The test requires that a known amount of O2 is brought in contact with a suitable amount of sample in such
a way that upon incubation oxidative biodegradation can take place and that the unused O2 can be
determined at the end of the incubation period. This is realized by introducing into a glass-stoppered bottle
a known amount of appropriately diluted sample after which the bottle is completely filled with fully aerated
dilution water, which contains nutrient minerals as well as appropriate bacterial "seed" material when
required.
By using completely filled, glass-stoppered bottles the available O2 is restricted to O2 dissolved in the
water. The dissolved O2 concentration in the bottle is determined at the beginning and at the end of the

58
Oxygen

incubation period. As a rule different dilutions are used; tests showing a residual O2 concentration of at
least 1 mg O2/L and a depletion of at least 2 mg O2/L are considered to be most reliable.
As a rule, samples have to be strongly diluted to fit the test conditions. If we know that the sample is
anaerobic, and if air-saturated water is used, the initial O2 concentration in the bottle can be computed. In
all other cases the initial O2 concentration has to be determined. The O2 determination is carried out
according to the oxygen electrode method. (Alternatively, the azide modification of Winkler's titration
method can be used).
A blank bottle, filled with dilution water only, is included when "seed" material is used. The O2 consumed in
this bottle during incubation is due to endogenous respiration of the bacteria in the seed material and
hence a correction has to be made for this.

The BOD of wastewater will be determined according to the American Standard Methods. Dilution water
has been prepared by aerating distilled water at a temperature of approximately 20°C. Just before use,
MgSO4, CaCl2, FeCl3, and phosphate buffer have been added to it in the concentrations indicated in the
Standard Methods. In some cases, the dilution water must be inoculated ("seeded") with a washed
bacterial suspension.
Transfer 1 mL of the sample to your BOD bottle. Note the volume of your bottle (around 300 mL). The
bottles are then filled to completion with dilution water and closed, taking care no air bubbles remain. The
initial O2 concentration can be calculated since an anaerobic sample solution will be used. In this case,
however, the volume of the added sample is so small it can be neglected. Hence the O2 found in the blank
bottle at the end of the incubation period can be taken as initial O2, which then automatically includes the
correction for the endogenous respiration of the added "seed" bacteria. A water-seal is used to prevent O2
from the air from entering the bottle during incubation. This is achieved by placing the bottles in an inverted
position in a water bath. A blank, consisting of seeded dilution water only, is included.
Incubate at 20°C during 5 days.

5.2.3 Carbonaceous Versus Nitrogenous BOD

Oxidation of reduced forms of nitrogen, mediated by micro-organisms, exerts nitrogenous


demand. Nitrogenous demand historically has been considered an interference in the
determination of BOD, as clearly evidenced by the inclusion of ammonia in the dilution
water. The interference from nitrogenous demand can now be prevented by an inhibitory
chemical. If an inhibiting chemical is not used, the oxygen demand measured is the
sum of carbonaceous and nitrogenous demands!!

Measurement that include nitrogenous demand generally are not useful for assessing the
oxygen demand associated with organic material.
The extent of oxidation of nitrogenous compounds during the 5-d incubation period depends
on the presence of micro-organisms capable of carrying out this oxidation. Such organisms
usually are not present in raw sewage or primary effluent in sufficient numbers to oxidize
significant quantities of reduced nitrogen forms in the 5-d BOD test. Many biological
treatment plant effluents contain significant numbers of nitrifying organisms. Because
oxidation of nitrogenous compounds can occur in such samples, inhibition of nitrification is
recommended for samples of secondary effluent, for samples seeded with secondary
effluent, and for samples of polluted waters. Report results as CBOD5 when inhibiting the
nitrogenous oxygen demand. When nitrification is not inhibited, report results as BOD5.

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Oxygen

5.2.4 General Discussion

a) Principle
The method consists of filling with sample, to overflowing, an airtight bottle of the
specified size and incubating it at the specified temperature for 5 d. Dissolved oxygen is
measured initially and after incubation, and the BOD is computed from the difference
between initial and final DO. Because the initial DO is determined immediately after the
dilution is made, all oxygen uptake, including that occurring during the first 15 min. is
included in the BOD measurement.
b) Sampling and storage
Samples for BOD analysis may degrade significantly during storage between collection
and analysis, resulting in low BOD values. Minimize reduction of BOD by analyzing
sample promptly or by cooling it to near-freezing temperature during storage. However,
even at low temperature, keep holding time to a minimum.
Warm chilled samples to 20°C before analysis.
1) Grab samples
If analysis is begun within 2 h of collection, cold storage is unneccessary. If analysis
is not started within 2 h of sample collection, keep sample at or below 4°C from the
time of collection. Begin analysis within 6 h of collection; when this is not possible
because the sampling site is distant from the laboratory, store at or below 4°C and
report length and temperature of storage with the results. In no case start analysis
more than 24 h after grab sample collection. When samples are to be used for
regulatory purposes make every effort to deliver samples for analysis within 6 h of
collection

2) Composite samples
Keep samples at or below 4°C during compositing. Limit compositing period to 24 h.
Use the same criteria as for storage of grab samples, starting the measurement of
holding time from end of compositing period. State storage time and conditions as
part of the results.

5.2.5 Apparatus

a) Incubation bottles
250 to 300 mL capacity. Clean bottles with a detergent, rinse thoroughly and drain
before use. As a precaution against drawing air into the dilution bottle during incubation,
use a water-seal. Obtain satisfactory water seals by inverting bottles in a water bath or
by adding water to the flared mouth of special BOD bottles. Place a paper or plastic cup
or foil cap over flared mouth of bottle to reduce evaporation of the water seal during
incubation

b) Air incubator or water bath


Thermostatically controlled at 20 ± 1°C. Exclude all light to prevent possibility of
photosynthetic production of DO

5.2.6 Reagents

a) Phosphate buffer solution


Dissolve 0.85 g KH2PO4, 2.18 g K2HPO4, 2.22 g Na2HPO4.2H2O and 0.17 g NH4Cl in
about 50 mL distilled water and dilute to 100 mL. The pH should be 7.2 without further
adjustment. Discard reagent (or any of the following reagents) if there is any sign of
biological growth in the stock bottle
b) Magnesium sulfate solution
Dissolve 2.25 g MgSO4.7H2O in distilled water and dilute to 100 mL

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Oxygen

c) Calcium chloride solution


Dissolve 3.64 g CaCl2.2H2O in distilled water and dilute to 100 mL
d) Ferric chloride solution
Dissolve 0.025 g FeCl3.6H2O in distilled water and dilute to 100 mL
e) Acid and alkali solutions
1N for neutralization of caustic or acidic waste samples
1. Acid
Slowly and while stirring, add 28 mL conc sulfuric acid to distilled water.
Dilute to 1L.
2. Alkali
Dissolve 40 g sodium hydroxide in distilled water. Dilute to 1 L.

f) Sodium sulfite solution


Dissolve 1.575 g Na2SO3 in 1000 mL distilled water. This solution is not stable; prepare
daily
g) Nitrification inhibitor
2-chloro-6-(trichloromethyl) pyridine (TCMP)
h) Glucose-glutamic acid solution with BOD = 200
Dry reagent-grade glucose and reagent-grade glutamic acid at 103°C for 1 h. Add
150 mg glucose and 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare
fresh immediately before use
i) Ammonium chloride solution
Dissolve 1.15 g NH4Cl in about 500 mL distilled water, adjust pH to 7.2 with NaOH
solution and dilute to 1L. Solution contains 0.3 mg N/mL.

5.2.7 Procedure

a) Preparation of dilution water


Place disired volume of water in a suitable bottle and add 1 mL each of phosphate
buffer, MgSO4, CaCl2, and FeCl3 solutions/L of water. Seed dilution water, if desired, as
described in 5.2.7d. Test and store dilution water as described in 5.2.7b and c so that
water of assured quality always is on hand.
Before use bring dilution water temperature to 20°C. Saturate with DO by shaking in a
partially filled bottle or by aerating with organic-free filtered air. Alternatively, store in
cotton-plugged bottles long enough for water to become saturated with DO. Protect
water quality by using clean glassware, tubing and bottles.

b) Dilution water check


Use this procedure as a rough check on quality of dilution water.
If the oxygen depletion of a candidate water exceeds 0.2 mg/L obtain a satisfactory
water by improving purification or from another source. Alternatively, if nitrification
inhibition is used, store the dilution water, seeded as prescribed below, in a darkened
room at room temperature until the oxygen uptake is sufficiently reduced to meet the
dilution-water check criteria. Check quality of stored dilution water on use, but do not
add seed to dilution water stored for quality improvement. Storage is not recommended
when BODs are to be determined without nitrification inhibitions because nitrifying
organisms may develop during storage. Check stored dilution water to determine
whether sufficient ammonia remains after storage. If not, add ammonium chloride
solution to provide a total of 0.45 mg ammonia/L as nitrogen. If dilution water has not
been stored for quality improvement, add sufficient seeding material to produce a DO
uptake of 0.05 to 0.1 mg/L in 5 d at 20°C. Incubate a BOD bottle full of dilution water for
5 d at 20°C. Determine initial and final DO as in 5.2.7g and j. The DO uptake 5 d at
20°C should not be more than 0.2 mg/L and preferably not more than 0.1 mg/L

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c) Glucose-glutamic acid check


Because the BOD test is a bioassay its results can be influenced greatly by the
presence of toxicants or by use of a poor seeding material. Distilled waters frequently
are contaminated with copper; some sewage seeds are relatively inactive. Low results
always are obtained with such seeds and waters. Periodically check dilution water
quality, seed effectiveness, and analytical technique by making BOD measurements on
pure organic compounds and samples with known additions. In general, for BOD
determinations not requiring an adapted seed, use a mixture of 150 mg glucose/L and
150 mg glutamic acid/L as a "standard” check solution. Glucose has an exceptionally
high and variable oxidation rate but when it is used with glutamic acid, the oxidation rate
is stabilized and is similar to that obtained with many municipal wastes. Alternatively, if a
particular wastewater contains an identifiable major constituent that contributes to the
BOD, use this compound in place of the glucose-glutamic acid.
Determine the 5d 20°C BOD of a 2% dilution of the glucose-glutamic acid standard
check solution using the techniques outlined in 5.2.7d-j.

d) Seeding
1. Seed source
It is necessary to have present a population of microorganisms capable of oxidizing
the biodegradable organic matter in the sample. Domestic wastewater,
unchlorinated or otherwise-undisinfected effluents from biological waste treatment
plants, and surface waters receiving wastewater discharges contain satisfactory
microbial populations. Some samples do not contain a sufficient microbial population
(for example, some untreated industrial wastes, disinfected wastes, high-
temperature wastes, or wastes with extreme pH values). For such wastes seed the
dilution water by adding a population of microorganisms. The prefered seed is
effluent from a biological treatment system processing the waste. Where this is not
available, use supernatant from domestic wastewater after settling at room
temperature for at least 1 h but no longer than 36 h. When effluent from a biological
treatment process is used, inhibition of nitrification is recommended.
Some samples may contain materials not degraded at normal rates by the
microorganisms in settled domestic wastewater. Seed such samples with an
adapted microbial population obtained from the undisinfected effluent of a biological
process treating the waste. In the absence of such a facility, obtain seed from the
receiving water below (preferably 3 to 8 km) the point of discharge. When such seed
sources also are not available, develop an adapted seed in the laboratory by
continuously aerating a sample of settled domestic wastewater and adding small
daily increments of waste. Optionally use a soil suspension or activated sludge,
or a commercial seed preparation to obtain the initial microbial population.
Determine the existence of a satisfactory population by testing the performance of
the seed in BOD tests on the sample. BOD values that increase with time of
adaptation to a steady high value indicate succesful seed adaption.

2. Seed control
Determine BOD of the seeding material as for any other sample. This is the seed
control. From the value of the seed control and a knowlegde of the seeding material
dilution (in the dilution water) determine seed DO uptake. Ideally, make dilutions of
seed such that the largest quantity results in at least 50% DO depletion. A plot of DO
depletion, in milligrams per liter, versus milliliters seed should present a straight line
for which the slope indicates DO depletion per milliliter of seed. The DO-axis
intercept is oxygen depletion caused by the dilution water and should be less than
0.1 mg/L (5.2.7h). To determine a sample DO uptake subtract seed DO uptake from
total DO uptake. The DO uptake of seeded dilution water should be between 0.6 and
1.0 mg/L.
Techniques for adding seeding material to dilution water are described for two
sample dilution methods (5.2.7f)

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Oxygen

e) Sample pre-treatment
1. Samples containing caustic alkalinity or acidity:
Neutralize samples to pH 6.5 to 7.5 with a solution of sulfuric acid (H2SO4) or sodium
hydroxide (NaOH) of such strength that the quantity of reagent does not dilute the
sample by more than 0.5%. The pH of seeded dilution water should not be affected
by the lowest sample dilution.

2. Samples containing residual chlorine compound:


If possible, avoid samples containing residual chlorine by sampling ahead of
chlorination processes. If the sample has been chlorinated but not detectable
chlorine residual is present, seed the dilution water. If residual chlorine is present,
dechlorinate sample and seed the dilution water (5.2.7f). Do not test
chlorinated/dechlorinated samples without seeding the dilution water. In some
samples chlorine will dissipate within 1 to 2 h of standing in the light. This often
occurs during sample transport and handling. For samples in which chlorine residual
does not dissipate in a reasonably short time, destroy chlorine residual by adding
Na2SO3 solution.
Determine required volume of Na2SO3 solution on a 100 to 1000 mL portion of
neutralized sample by adding 10 mL of 1 + 1 acetic acid or 1 + 50 H2SO4, 10 mL
potassium iodide (KI) solution (10 g/100 mL) per 1000 mL portion, and titrating with
Na2SO3 solution to the starch-iodine end point.
Add to neutralized sample the relative volume of Na2SO3 solution determined by the
above test, mix, and after 10 to 20 min. check sample for residual chlorine.

NOTE: Excess Na2SO3 exerts an oxygen demand and reacts slowly with certain
organic chloramine compounds that may be present in chlorinated samples.

3. Samples containing other toxic substances


Certain industrial wastes, for example, plating wastes, contain toxic metals. Such
samples often require special study and treatment.

4. Samples supersaturated with DO


Samples containing more than 9 mg DO/L at 20°C may be encountered in cold
waters or in water where photosynthesis occurs. To prevent loss of oxygen during
incubation of such samples, reduce DO to saturation at 20°C by bringing sample to
about 20°C in partially filled bottle while agitating by vigorous shaking or by aerating
with clean, filtered compressed air.

5. Sample temperature adjustment


Bring sample to 20 ± 1°C before making dilutions

6. Nitrification inhibition
If nitrification inhibition is desired add 3 mg 2-chloro-6-(trichloromethyl) pyridine
(TCMP) to each 300 mL bottle before capping or add sufficient amounts to the
dilution water to make a final concentration of 10 mg/L.

NOTE: Pure TCMP may dissolve slowly and can float on top of the sample. Some
commercial formulations dissolve more readily but are not 100% TCMP; adjust
dosage accordingly.

Samples that may require nitrification inhibition include, but are not limited to,
biologically treated effluents, samples seeded with biologically treated effluents, and
river waters. Note the use of nitrogen inhibition in reporting results.

f) Dilution technique
Dilutions that result in a residual DO of at least 1 mg/L and a DO uptake of at least
2 mg/L after 5 d incubation produce the most reliable results. Make serveral dilutions of
prepared sample to obtain DO uptake in this range. Experience with a particular sample

63
Oxygen

will permit use of a smaller number of dilutions. A more rapid analysis, such as COD,
may be correlated approximately with BOD and serve as a guide in selecting dilutions.
In the absence of prior knowledge, use the following dilutions: 0.0 to 1,0% for strong
industrial wastes, 1 to 5% for raw and settled wastewater, 5 to 25% for biologically
treated effluent, and 25 to 100% for polluted river waters.

Prepare dilutions either in graduated cylinders and then transfer to BOD bottles or
prepare directly in BOD bottles. Either dilution method can be combined with any DO
measurement technique. The number of bottles to be prepared for each dilution
depends on the DO technique and the number of replicates desired.

When using graduated cylinders to prepare dilutions, and when seeding is necessary,
add seed either directly to dilution water or to individual cylinders before dilution.
Seeding of individual cylinders avoids a declining ratio of seed to sample as increasing
dilutions are made. When dilutions are prepared directly in BOD bottles and when
seeding is necessary, add seed directly to dilution water or directly to the BOD bottles.

1. Dilutions prepared in graduated cylinders


If the azide modification of the titrimetric iodometric method is used, carefully siphon
dilution water, seeded if necessary, into a 1 to 2 L-capacity graduated cylinder. Fill
cylinder half full without entraining air. Add desired quantity of carefully mixed
sample and dilute to appropriate level with dilution water. Mix well with a plunger-
type mixing rod; avoid entraining air. Siphon mixed dilution into two BOD bottles.
Determine initial DO on one of these bottles. Stopper the second bottle tightly,
water-seal, and incubate for 5 d at 20°C. If the membrane electrode method is used
for DO measurement, siphon dilution mixture into one BOD bottle. Determine initial
DO on this bottle and replace any displaced contents with sample dilution to fill the
bottle. Stopper tightly, water-seal, and incubate for 5 d at 20°C.

2. Dilutions prepared directly in BOD bottles


Using a widetip volumetric pipet, add the desired sample volume to individual BOD
bottles of known capacity. Add appropriate amounts of seed material to the
individual BOD bottles or to the dilution water. Fill bottles with enough dilution water,
seeded if necessary, so that insertion of stopper will displace all air, leaving no
bubbles. For dilutions greater than 1:100 make a primary dilution in a graduated
cylinder before making final dilution in the bottle. When using titrimetric iodometric
methods for DO measurement, prepare two bottles at each dilution. Determine initial
DO on one bottle. Stopper second bottle tighly, water-seal, and incubate for 5 d at
20°C. If the membrane electrode method is used for DO measurement, prepare only
one BOD bottle for each dilution. Determine initial DO on this bottle and replace any
displaced contents with dilution water to fill the bottle. Stopper tightly, water-seal,
and incubate for 5 d at 20°C. Rinse DO electrode between determinations to prevent
cross-contamination of samples.

g) Determination of initial DO
If the sample contains materials that react rapidly with DO, determine initial DO
immediately after filling BOD bottle with diluted sample. If rapid initial DO uptake is
insignificant, the time period between preparing dilution and measuring initial DO is not
critical.
Use the azide modification of the iodometric method or the membrane electrode method
to determine initial DO on all sample dilutions, dilution water blanks, and where
appropriate, seed controls.

h) Dilution water blank


Use a dilution water blank as a rough check on quality of unseeded dilution water and
cleanliness of incubation bottles. Together with each batch of samples incubate a bottle
of unseeded dilution water. Determine initial and final DO als in 5.2.7g and j. The DO
uptake should not be more than 0.2 mg/L and preferably not more than 0.1 mg/L.

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Oxygen

i) Incubation
Incubate at 20°C ± 1°C BOD bottles containing desired dilutions, seed controls, dilution
water blanks, and glucose-glutamic acid checks. Water-seal bottles as described in
5.2.5a.

j) Determination of final DO
After 5 d incubation determine DO in sample dilutions, and checks as in 5.2.7g.

5.2.8 Calculation

Calculate from this result the BOD520 of the wastewater. The calculations can performed as
follows:

Suppose Na2S2O3 consumption for blank bottle and sample bottle is b ml and s ml, respectively.
This corresponds with b * M * 8 mg O2 and s * M * 8 mg O2, respectively.

b * M *8 s * M *8
Or mg O2/mL and mg O2/mL, respectively
Vb − 2 Vs − 2
(-2, because of 2 ml reagent introduced).
Due to bio-oxidation there will be consumed

( b * M *8 s * M *8
− ) mg O2/mL or ( b

s
) * M * 8 mg O2/mL.
Vb − 2 Vs − 2 V b−2 Vs − 2

If the sample bottle has been filled with c ml sample and, for the rest dilution water, there will be
required:

Vs
( b

s
)* M *8* mg O2/mL or
V b−2 Vs − 2 c

Vs
( b

s
)* M *8* * 1000 mg O2/L which is the BOD520.
V b−2 Vs − 2 c

Vb = volume blank bottle; Vs = volume sample bottle;


M = molarity of standard Na2S2O3 solution; d = dilution factor for sample.

Report results as CBOD5 if nitrification is inhibited.


If more than one sample dilution meets the criteria of a residual DO of at least 1 mg/L and a
DO depletion of at least 2 mg/L and there is no evidence of toxicity at higher sample
concentrations or the existence of an obvious anomaly, average results in the acceptable
range.

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Oxygen

5.2.9 The oxygen electrode method

Measure the oxygen concentration with an oxygen electrode, immediately at the start of the
experiment and after 5 days. The DO concentration in the sample bottle after 5 days should
be above 1.0 mg/L, but at least 2.0 mg/L oxygen should be consumed.
The DO consumption of the blank should preferably be not more than 0.2 mg/L.

When dilution water is not seeded:

( So − Sv ) * Vs
BOD5 (in mg/L) =
c

And when dilution water is seeded

( So − Sv ) − ( Bo − Bv ) * f * Vs
BOD5 (in mg/L) =
c

So = DO sample, immediately at the start (mg/L)


Sv = DO sample after 5 days (mg/L)
Bo = DO blank, immediately at the start (mg/L)
Bv = DO blank after 5 days (mg/L)
f = ratio of seed in diluted sample to seed in seed control =
(% seed in diluted sample)/(% seed in seed control)
Vs = volume of sample bottle (mL)
c = volume of sample (mL)

When the sample volume is just a few mL (in case of samples with a high BOD) the following
formula can be used as well:

( B − S ) *Vb
BOD = = mg /L
c
B = Dissolved Oxygen blank after 5 days (mg/L)
S = Dissolved Oxygen sample after 5 days (mg/L)
Vb = Volume sample-bottle (mL)
c = Volume sample (mL)

Remarks:
• It is important to use a good, well-calibrated DO-probe
• No gas bubbles should be collected under the probe when placing the probe into the
bottle.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 5-2/5-6.

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5.3 Chemical Oxygen Demand (COD)

5.3.1 Introduction

The COD is used as a measure of the oxygen equivalent of the organic matter content of a
sample that is susceptible to oxidation by a strong chemical oxidant.
Oxidation of most organic compounds is 95-100% of the theoretical value. Pyridine and
related compounds resist oxidation and volatile organic compounds are oxidized only to the
extent that they remain in contact with the oxidant.
COD analysis will never yield 100% of the stochiometric ThOD calculations as the oxidation
of reduced nitrogen is not included in the standard COD test.
Ammonia, present either in the waste or liberated from nitrogen-containing organic matter, is
not oxidized in the absence of significant concentration of free chloride ions. In this
procedure these chloride ions are complexed with HgSO4.

5.3.2 Interferences and limitations

Do not use the test for samples containing more than 2000 mg Cl- /L. Techniques designed
to measure COD in saline waters are available. NO - and reduced inorganic species such
2
as Fe2+, S2-, M42+ etc. are oxidized quantitatively. To eliminate a significant interference due
to NO - sulfamic acid can be added.
2
Ag acts as a catalyst and forms precipitates with halides.

5.3.3 Sampling and storage

Preferably collect samples in glass bottles. Test unstable samples without delay, if delay is
unavoidable, preserve sample by acidification to pH ≤ 2 using conc. H2SO4.

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5.3.4 Open Reflux, Titrimetric Method

5.3.4.1 Principle

Most types of organic matter are oxidized by a boiling mixture of chromic and sulfuric acids.
A sample is refluxed in strongly acid solution with a known excess of K2Cr2O7. After
digestion, the remaining unreduced K2Cr2O7 is titrated with ferrous ammonium sulfate (FAS)
to determine the amount of K2Cr2O7 consumed and the oxidazable organic matter is
calculated in terms of oxygen equivalent.

5.3.4.2 Apparatus

Reflux apparatus.

5.3.4.3 Reagents

a) K2Cr2O7 0.0417 M: Dissolve 12.259 g K2Cr2O7, previously dried at 103°C for 2 hours,
in 1000 mL H2O
b) H2SO4 - reagent: Add 10 g Ag2SO4 to 1 L conc. H2SO4, let stand overnight to
dissolve. Mix carefully after dissolving
c) Ferroin indicator: Dissolve 1.485 g 1,10 phenanthroline monohydrochloride
monohydrate and 695 mg FeSO4.7H2O in H2O and dilute to 100
mL
d) FAS titrant 0.1 M: Add 20 mL conc. H2SO4 to 1 L H2O, mix and dissolve
40 g (NH4)2.Fe(SO4)2.6H2O
e) HgSO4
f) Potassium hydrogen phthalate
(KHP) standard: Lightly crush and then dry KHP (HOOCC6H4COOK) to constant
weight at 120°C. Dissolve 425 mg per liter water. This solution has
a theoretical COD of 500 mg O2/L. This solution is stable when
refrigerated for up to 3 months in the absence of visible biological
growth.

FAS standardization (Use safety spectacles)

1) Dilute 10.00 mL (pipet) of the standard K2Cr2O7 solution to about 100 mL (erlenmeyer
flask)
2) Carefully add 30 mL concentrated H2SO4 with a measuring cylinder. Mix well. Cool
down to ca. 20°C
3) Add 4 drops ferroin indicator
4) Fill a buret with FAS solution
5) Titrate with the FAS solution until the colour changes from blue-green to reddisch-
brown.
2 .5
Molarity FAS =
mL FAS

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Oxygen

5.3.4.4 Procedure for 50<COD<900

Because of the corrosive substances involved, it is necessary to wear safety


spectacles and, preferably, plastic gloves! In case of spilling on your hands, wipe off
the acid, then rinse extensively under (cool) tapwater immediately.

1. Transfer about 20 pumice grains to the reflux flask


2. Add about 0.4 g HgSO4
3. With a pipet, add 20.00 mL of the sample and/or standard KHP and mix. Also carry out
a blank determination, using 20.00 mL of demineralized water
4. With a pipet, add 10.00 mL 0.0417 M (0.25N) K2Cr2O7 solution and mix
5. With a measuring cylinder add very slowly, with mixing, about 30 mL sulfuric acid
reagent
6. Mix until the solution is completely homogeneous
7. Attach the reflux flask to the condenser, turn on the cooling water, light the gas burner
and reflux gently for exactly two hours
8. Wait 5-10 minutes for cooling-off, then wash down the condenser with about 50 mL
demineralized water.

Titration of excess dichromate:

9. Fill a buret with standard FAS solution


10. Cool the reflux flask to room temperature under a tap. Mix well
11. Add 4 drops ferroin indicator
12. Titrate with standard FAS solution until the colour changes from blue-green to reddish-
brown. The blue-green colour may reappear on standing, but this should be ignored

Calculation:

( A − B ) x M x 8000
COD as mg O2/L = , where
mL sample

A = mL FAS used for blank


B = mL FAS used for sample
M = molarity of FAS

Remark: If COD>900 repeat determination with diluted sample, if COD<50 see


procedure 5.3.4.5

5.3.4.5 Alternate procedure for COD<50

Follow procedure 5.3.4.4 with two exceptions:

1) Use standard 0.00417 M K2Cr2O7 and


2) Titrate with 10 times dilluted FAS (about 0.01 M).

Exercise extreme care with this procedure because even a trace of organic matter on the
glassware or from the atmosphere may cause gross errors.

5.3.4.6 Determination of standard solution

Evaluate the technique and quality of reagents by conducting the test on a standard KHP
solution.

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5.3.5 Closed Reflux, Colorimetric Method

5.3.5.1 Apparatus

Digestion vessels 15 x 180 mm with screw caps.


Oven to operate at 150 ± 2°C.
Spectrophotometer for use at 600 nm

5.3.5.2 Reagents

a) Digestion solution: Add to about 500 mL H2O 10.216 g K2Cr2O7, previously dried at
103°C for 2 hours, 167 mL conc. H2SO4 and 33.3 g HgSO4. Dissolve
(this will take many hours, mixing during this time is not required,
however mix after dissolving), cool to room temperature and dilute to
1000 mL

b) H2SO4/Ag2SO4: Add 10 g Ag2SO4 to 1 L conc. H2SO4, let stand overnight to dissolve.


Mix carefully after dissolving

c) Stock KHP: 850 mg potassium hydrogen phthalate, dried at 120°C for 24 hours, is
dissolved in 1000 mL H2O.
The COD of this stock solution is 1000 mg O2/L.

5.3.5.3 Preparation of standards

Prepare standard solutions containing a known COD concentration by diluting a known


volume, A, of the stock KHP to 100 mL.

mL A/100 mL COD mg/L

0 0
2 20
5 50
7 70
10 100
20 200
30 300
40 400
50 500
60 600
70 700
80 800
90 900

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Oxygen

5.3.5.4 Procedure

Wash the digestion tubes and caps with 4 M H2SO4 before first use to prevent
contamination.

1. Transfer 2.5 mL of standard or sample to the digestion tube and add 1.5 mL digestion
solution
2. Carefully run 3.5 mL H2SO4/Ag2SO4 down inside of tube so an acid layer is formed
under the sample-digestion solution layer. Tightly cap tubes and swirl several times to
mix completely, do not invert the tubes!
3. Place the tubes in a preheated oven of 150°C during 2 hours
4. Allow them to cool, mix the content and let particles settle

5. The next day:


Transfer the content gently and without mixing to a 1 cm cell and measure the
absorbance at 600 nm against water
6. Plot the absorbance against the known COD in order to get a calibration line
7. Read absorbance of samples and compare to calibration line, determine the
mathematical equation of this line.

Remarks:
1. Use an adjustable Eppendorf pipette
2. Run the standard and samples in duplicate
3. For each set of samples also analyse simultaneously one or two standards with a COD
value comparable to the samples.

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 5-6/5-11.

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5.4 Total Organic Carbon (TOC)

5.4.1 General Discussion

The organic carbon in water and wastewater is composed of a variety of organic compounds
in various oxidation states. Some of these carbon compounds can be oxidized further by
biological or chemical processes, and the biochemical oxygen demand (BOD) and chemical
oxygen demand (COD) may be used to characterize these fractions. The presence of
organic carbon that does not respond to either the BOD of COD test makes them unsuitable
for the measurement of total organic carbon. Total organic carbon (TOC) is a more
convenient and direct expression of total organic content than either BOD or COD, but does
not provide the same kind of information. If a repeatable empirical relationship is established
between TOC and BOD or COD, then TOC can be used to estimate the accompanying BOD
or COD.
This relationship must be established independently for each set of matrix conditions, such
as various points in a treatment process. Unlike BOD or COD, TOC is independent of the
oxidation state of the organic matter and does not measure other organically bound
elements, such as nitrogen and hydrogen, and inorganics that can contribute to the oxygen
demand measured by BOD and COD. TOC measurement does not replace BOD and COD
testing.

To determine the quantity of organically bound carbon, the organic molecules must be
broken down to single carbon untis and converted to a single molecular form that can be
measured quantitatively. TOC methods utilize heat and oxygen, ultraviolet irradiation,
chemical oxidants, or combinations of these oxidants to convert organic carbon to carbon
dioxide (CO2). The CO2 may be measured directly by a nondispersive infrared analyzer, it
may be reduced to methane and measured with a flame ionization detector or CO2 may be
titrated chemically.

5.4.2 Fractions of Total Carbon

The methods and instruments used in measuring TOC analyze fractions of total carbon (TC)
and measure TOC by two or more determinations. These fractions of total carbon are
defined as:
• inorganic carbon (IC)
the carbonate, bicarbonate, and dissolved CO2;
• total organic carbon (TOC)
all carbon atoms covalently bonded in organic molecules;
• dissolved organic carbon (DOC)
the fraction of TOC that passes through a 0.45-μm-pore-diam filter;
• particulate organic carbon (POC)
also referred to as nondissolved organic carbon, the fraction of TOC retained by a
0.45 μm filter;
• volatile organic carbon (VOC)
also referred to as purgeable organic carbon, the fraction of TOC removed from an
aqueous solution by gas stripping under specified conditions;
• nonpurgeable organic carbon (NPOC)
the fraction of TOC not removed by gas stripping.

In most water samples, the IC fraction is many times greater than the TOC fraction.
Eliminating or compensating for IC interferences requires multiple determinations to
measure true TOC. IC interference can be eliminated by acidifying samples to pH 2 or less
to convert IC species to CO2.

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Oxygen

Subsequently, purging the sample with a purified gas removes the CO2 by volatilization.
Sample purging also removes VOC so that the organic carbon measurement made after
eliminating IC interferences is actually a NPOC determination; determine VOC to measure
true TOC.

In many surface and ground waters the VOC contribution to TOC is negligible. Therefore, in
practice, the NPOC determination is substituted for TOC.
Alternatively, IC interference may be compensated for by separately measuring
inorganic carbon (IC) and total organic carbon (TOC).

Literature:
American Public Health Organisation
Standard Methods for the examiniation of water and wastewater, 18th edition 1992
p. 5-10/5-15.

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Miscellaneous

6. MISCELLANEOUS

6.1 Biomass

6.1.1 Biomass according to White

Principle:

This determination is based on the fact that phospholipids are present in almost all
membranes in a relatively constant ratio. In living cells as well as in dead cells the turnover
of phospholipids is relatively high. Because of this fact they are suitable as a measure for the
amount of living biomass in a sample (For bacillus-like bacteria it is assumed that 1 gram of
dry cells contain 50 μmol of lipidphosphate).

The determination consists of three parts:


- extraction of lipids
- destruction of lipids
- determination of phosphate

6.1.2 Apparatus and glassware

a) Shaking machine
b) Oven, adjustable till 180°C
c) Separation funnels of 250 mL, acid rinsed and washed with water
d) Destruction tubes, acid rinsed. After acid treatment, boiled with water and dried without
touching the inner parts
e) Stoppered 250 mL glass bottles, acid rinsed and washed with water. Idem for several
pipettes
f) Marbles, acid rinsed and washed with water
g) Spectrophotometer for use at 830 nm with 1 cm cells
h) 100 mL volumetric flasks, acid rinsed and washed with water
i) Waterbath at 100°C.

6.1.3 Reagents

a) CH3OH, methanol
b) CHCl3, chloroform
c) HClO4, perchloric acid, 60% and 30%
d) H2O2, hydrogenperoxide, 30%

e) Stock β-glycerol-phosphate solution (C3H7O6PNa2.4H2O M=288) of 250 mg/L


f) Ammoniumheptamolybdate reagent: Dissolve 5 g (NH4)6Mo7O24.4H2O in 100 mL.
Store at 4°C
g) ANSA reagent: Dissolve 45 g NaHSO3, 2.1 g Na2SO3 and 0.5 g ANSA
(1-amino-2-naphtol-4-sulfonic acid M=239.25) in 100 mL

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Miscellaneous

Let stabilize for 1 night at 4°C and put the clear liquid in a dark bottle.
6.1.4 Calibration

1. Prepare a serie of standards by diluting 0; 10.00; 30.00; 50.00 and 80.00 mL stock to
100 mL in volumetric flasks and fill up to the mark
2. Place 0.50 mL of each standard in a destruction tube and add 0.50 mL 60% HClO4.
Follow from here on the destruction procedure 6.1.6 starting with step 4 and further
3. Plot the absorbance against nmol P and determine the mathematical expression of this
line (400 nmol should give an absorption of about 2).

6.1.5 Extraction procedure for lipids

1. Weigh maximal 6 g dry weight sample and put in a glass stoppered bottle of 250 mL
(Perform dry-weight measurement separately if necessary)
2. Add 50 mL CH3OH, 25 mL CHCl3 and 20 mL H2O. Take into account the water present in
the sample
3. Close the bottle and shake at room temperature during 2 h
4. Add 25 mL CHCl3 and 25 mL H2O

5. Mix thoroughly
6. Transfer the content to a separation funnel and leave the sample for 24 h (overnight) at
4°C for de-mixing
7. Collect a few mL of the lowest (CHCl3) layer without water. If necessary, centrifuge this
layer
8. If necessary, store at 4°C (stable for at least 1 week).

6.1.6 Destruction procedure for lipids

1. Take a sample from the chloroform layer, e.g. 0.50 mL, and transfer to a destruction tube
2. Evaporate the CHCl3 under a stream of air
3. Add 1.00 mL 30% HClO4

4. Mix well (vortex)


5. Cover the tube with a marble and place in an oven at 180°C during 20 minutes
6. When the destruction samples are not colourless, cool and add 0.1 mL H2O2 and heat
again at 180°C for 20 minutes. Repeat if necessary.

6.1.7 P-determination

1. Add 4.50 mL H2O to the destruction tubes with sample/standard

2. Add 0.25 mL molybdate reagent and 0.25 mL ANSA reagent


3. Mix well (vortex) and place in a waterbath of 100°C for 7 minutes, keep the marble on top
4. Cool, mix and measure the absorbance at 830 nm against H2O.

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Miscellaneous

6.1.8 Calculation

nmol ( from graph ) x 50


μmol P/g dry weight =
mL sample ( CHCl 3 layer ) x g extracted dry weight x 1000

6.1.9 Remark

Flush afterwards the destruction tubes 3 x with H2O and store the tubes and the marbles in
H2O.

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Miscellaneous

6.2 Suspended Solids, (SS or dry weight) and Volatile Suspended Solids
(VSS or ash-free dry weight)

6.2.1 Materials

a) Suction apparatures
b) GF/C filters or GF52 filters
c) Oven, 105ºC
d) Muffle furnace, 520ºC
e) Aluminium cups

6.2.2 Procedure

1. Put the filter in a cup and heat for 2 hours at 520ºC


This removes any organic material in the filter
2. Store cup + filter in the oven at 105ºC or in the dessiccator
3. Determine, after cooling, the clean weight (= cw) of filter + cup

4. Filter water sample and return filter TO THE SAME CUP


5. Dry for 2 hours at 105ºC
6. Determine dry weight of sample + filter + cup (= dwc)
7. SS, (dry weight) = dwc - cw

8. Remove sample + filter from cup and weigh the sample + filter (= a)
9. Put sample + filter back in the cup and heat at 520ºC for 3 hours
10. Cool in the dessiccator and weigh sample + filter (= b)
11. VSS, (ash-free dry weight) = a - b

Remark:
Since the weight of Al-cups will change after treatment at 520ºC, it is important to carry out
the weighing procedures from step 8 on without the Al-cups.

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Miscellaneous

6.3 Chlorophyll-a in algae

6.3.1 Principle

Algae will be filtered and extracted with ethanol. The difference in absorbance of the solution
before and after acidification, measured at 665 and 750 nm is a measure of the chlorophyll-a
content. Degradation products of chlorophyll-a show absorption spectra that are comparable
to the one of chlorophyll-a, and thus will result in an overestimation. By acidification of a
sample, all chlorophyll present is transformed into phaeophytine. Thus, by subtracting the
absorption values after acidification from the ones measured before acidification, the
absorption due to degradation products can be measured. The measurements at 750 nm
are necessary to correct for the turbidity.

6.3.2 Sampling and storage

Transport the sample in a dark bottle preferably at 4ºC, filter the algae as soon as possible
after sampling using glass fiber or membrane (0.45 µm porosity) filter. The sample can be
stored no longer than 24 h at 4ºC. The filter with algae can be stored at -18ºC or lower
during 2 weeks in the dark.

6.3.3 Apparatus and materials

a) Waterbath with the possibility to shake


b) Centrifuge
c) Spectrophotometer with 10 or 50 mm cells for measurements at 665 and 750 nm
d) Schleicher and Schuell GF6 filters (99.9% efficiency for > 0.3 μm)
[or Whatman GF/F (98 % for > 0.7 µm or Whatman GF/B (98 % for > 1.0 µm)]
e) Schott GL18 COD tubes

6.3.4 Reagents

a) Ethanol 80% (v/v): Dilute 833 mL 96% ethanol to 1000 mL


b) HCl 0.4 M: Dilute 10 mL 12 M HCl (1.19 g/mL) to 300 mL

6.3.5 Procedure

Choose a sample volume, normally between 50-2000 mL, that will result in an absorbance
between 0.1 and 0.9 at 665 nm for the non-acidified extract.
Carry out the following steps under reduced light intensity.

1. Filter a sample volume (Vo) using a GF6 filter

2. Transfer the filter to a Schott GL18 COD tube, add 25.0 mL 80% ethanol; close the tube
3. Put the tube in a waterbath of 75ºC and leave it there for 5 minutes. Use a rod to promote
the destruction
4. Shake the tube during 5 min. at 75ºC with a speed of 250 rpm
5. Cool the tube in ice
6. Divide the 25 mL extracts into centrifuge tubes and centrifuge the extract for 10 minutes
at 3000 rpm, let it stop by itself without using the brake

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Miscellaneous

7. Combine the extracts, mix and measure the absorbance at 750 nm (Eo) and 665 nm (Ex)
against 80% ethanol
8. Add 0.10 mL 0.4 M HCl to 10.0 mL extract and measure after 5-30 min again at 750 nm
(Eoa) and 665 nm (Exa) against 80% ethanol. The pH of this extract should be 2.6-2.8

6.3.6 Calculation

The corrected absorbance of the non-acidified extract : En = Ex-Eo

The corrected absorbance of the acidified extract : Ea = Exa - Eoa

En - Ea
Chl-a μg/L = 296 x V1 x , where
Vo x p

296 = factor based on the specific absorption coefficient of Chl-a


V1 = volume of 80% ethanol added to the filter with algae, in mL (25)
Vo = sample volume which is filtered, in L
p = path length of the cuvette in mm

Literature:
Nederlandse Norm (Dutch Standard) NEN 6520.

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Miscellaneous

6.3.7 Chlorophyll-a in macrophytes

6.3.7.1 Principle

Measuring of chlorophyll a and b and their decomposition products (phaeopigments) is done


according to Wintermans & De Mots (1965). Firstly, chlorophyll a and b is measured at
750 nm (blank), 665 nm (chlor a) and 649 nm (chlor b).
Then the sample is diluted with 0.06 M HCl. This acidification step makes the chlorophyll to
decompose in phaeopigments. Readings are now at different wavelenghts (666 and 655 nm)
because spectrum has moved due to acidification. Each sample is so measured at 5
different wavelenghts ( 750 nm, 665 nm, 649 nm, 666 nm and 655 nm).

6.3.7.2 Reagents and materials

measuring cylinder of 10 mL
mortar and pestle
screw-capped centrifuge tubes marked at 5 mL
tube rack
measuring pipets of 1.0 and 5.0 mL
96% ethanol
0.06 M HCl

6.3.7.3 Procedure

During extraction you should work at dimlight conditions to prevent chlorophyll


decomposition by light.

1. Set aside 5 mL 96% ethanol in a measuring cylinder for each sample


2. Grind 0.1-0.3 g fresh weight of plantmaterial in a mortar with 2 mL ethanol (Preserved
frozen samples are easier to grind!)
3. Grind until a green suspension is about all that is left of the plantmaterial
4. Transfer the suspension quantitatively to a screw-capped centrifuge tube
5. Use the remaining 3 mL of ethanol to rinse off the pestle and mortar and transport this
solution also as much as possible to the tube
6. Fill up the tube with ethanol to the 5 mL mark on the centrifuge tube
7. Put a cap on the tube to prevent evaporation of the ethanol
8. Leave the sample overnight or at least for 12 hours in the dark at roomtemperature
for maximum extraction
9. The next day:
Centrifuge for about 10 minutes at 3000 rpm, there should be a clear supernatant left
Keep the samples as dark as possible
10. Zero the apparatus with ethanol at all wavelengths
11. Carefully pipet 3 mL of the supernatant into a cuvet
12. Measure at 750, 665 and 649 nm
13. Add 0.5 mL of 0.06 M HCl to the cuvet
14. Measure at 750, 666 and 655 nm.

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Miscellaneous

6.3.7.4 Calculations

chlorophyll-a = 13.70 * (A665-A750) - 5.76 * (A649 - A750)


chlorophyll-b = 25.80 * (A649 - A750) - 7.60 * (A665 - A750)
total phaeopigments-a = 24.50 * (A666 - A750a) - 9.32 * (A665 - A750a)
total phaeopigments-b = 36.97 * (A665 - A750a) - 18.48 * (A666 - A750a)

where A750a = absorbance at 750 nm after acidification

The formula for chlorophyll-a expressed in mg/g of leaf:

[13 .70 * ( A665 − A750 ) − 5 .76 * ( A649 − A750 )] * Ve * D * C


Chl-a =
AFDW of sample * 1000

The same type of calculation can also be applied for the chlorophyll-b and
total-phaeopigments determination.

Remarks:
- In these basic formulae there is no correction for extraction volume (Ve) and sample
dilution (D)
- There will be an extra dilutionfactor (C) after acidification (adding 0.5 mL to 3 mL sample
in cuvette = 3.5/3); before acidification: C = 1
- Furthermore, [chlor] is given as ash-free dry weight (AFDW) so there should also be a
conversion from sample fresh weight (FWs):
AFDWs = FWs*(AFDW/FW)
- Phaeopigments-a in sample = (total phaeopigments-a) - (chlorophyll-a). This results in an
indication for planthealth, where a result close to zero stands for a very healthy plant.
Small amounts of phaeopigments-a means little decomposition of chlorophyll-a in the
sample.

Literature:
Wintermans, J.F.G.M. & De Mots A., 1965. Spectrophotometric characteristics of
chlorophylls a and b and their pheophytins in ethanol. Biochim. Biophys. Acta 109:448-453.

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Miscellaneous

6.4 Polycyclic Aromatic Hydrocarbons (PAH)

General

• Store the soil samples not longer than 4 days at 4ºC (in the dark)
• Soil samples must be dried at low temperature
• Cool the sample, after drying, with liquid N2 (-196ºC)
• Grind the sample
• Do the extraction immediately after grinding.

Reagents

• Petroleumether
• Na2SO4, heat for 6 hours at 500ºC, store in a dessiccator and cool before use to 4ºC
• Seasand, wash two times with an equal volume of water and heat for 6 hours at 500ºC
• Aluminiumoxide, deactivated, see next page
• Baby-powder (talkpoeder)

Sample treatment

• Add 20 g Na2SO4 and 5 g babypowder to a glass bottle with PTFE foil and screw cap
• Determine the mass of these reagents with an accuracy of 0.1 g
• Shake (mix) and cool to 4ºC
• Add about 25 g soil (determine the mass) to the bottle
• Shake well, place the bottle at 4ºC and shake during 4 hours
• Afterwards, let the bottle stand for 12-16 hours

Remark:
If necessary, add more Na2SO4
If big particles are present (>3 cm): crush with a spatula

• Mix the content again before grinding


• Transfer the content to a polyethylene bottle and cool with liquid N2 or, as alternative,
cool in the deepfreezer for 12 hours to about -20°C
• Grind the whole sample or a sub-sample. Keep as cool as possible
• Particles must be <2 mm
• Determine also the dry weight content of the original sample without Na2SO4 and
babypowder
• Follow the same procedure for the blank, use for this purpuse seasand

Analysis

• The efficiency of the procedure depends of the composition of the soil and the detention
time of the compounds in the soil.
The extraction efficiency depends also of the structure and composition of the soil

Safety

• Prevent any contact between skin and extracts or standards of the PAH's. Use plastic
gloves all the time!
Make use of the fume cupboard as much as possible!

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Miscellaneous

Deactivation procedure of aluminium oxide

• Dry aluminium oxide W 200, basic or neutral, activity Super I Woelm, during 16 hours at
150°C
• Cool in the dessiccator and add 11 g water to 89 g Al2O3
• Shake till there are no big particles left anymore
• Keep the deactivated Al2O3 for at least 16 hours in an air-tight vessel before use
• Store the deactivated Al2O3 always in an air-tight bottle.

Extraction and concentration

• Weight about 20 g sample with an accuracy of 0.1 g in a 300 mL erlenmeyer with glass
stopper
• Add 100 mL petroleumether and shake during 10 minutes on a shaking machine (linear
movement)
• Let the phases separate and transfer the petroleumether to the Kuderna Danish
equipment, use a funnel with glasswool
• Repeat the extraction with another 100 mL petroleumether, add this extract also to the
Kuderna Danish
• Rinse the erlenmeyer two times with 10 mL petroleumether, add this also to the Kuderna
Danish
• Evaporate the extract to a volume of about 10 mL, using the Kuderna Danish and a
waterbath of 70-75°C. Remove the tube and evaporate to a volume smaller than 1 mL,
use a gentle stream of N2 at room temperature. Adjust the height and the flow of the N2
inlet in such a way that it takes at least 15 minutes. Add some petroleumether till the
volume is 1.00 mL.

Purification of the extract (= clean up)

• Prepare an adsorption column by putting some glasswool in a chromatography tube and


add 2.0 ± 0.1 g deactivated Al2O3
• Put a Na2SO4 layer on top
• Transfer the extract by means of a pipet to the adsorption column. Rinse the K.D. tube
two times with 1 mL petroleumether and transfer this also to the column at the moment
that the liquid has penetrated the column. Elute with about 12 mL petroleumether. Collect
the eluate in a calibrated tube of 20 mL. Evaporate again with N2 at room temperature to
an endvolume of 1.00 mL. Transfer this extract to autosampler bottles with screw cap.
Store in the refrigerator before GC-analyses.

Chromatography tube

Literature:
Dutch Standard NVN 5730 Soil-Sample pretreatment for determination of organic
parameters in soil
Dutch Standard NEN 5731 Soil-Determination of ten polycyclic aromatic hydrocarbons with
HPLC.

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6.5 Cation Exchange Capacity of soil (CEC)

6.5.1 General

The theory on the cation exchange capacity of soils may directly be related to the theories
for adsorption isotherms and for ion exchange. The CEC of a soil is defined as the capacity
to bind and exchange cations. The CEC is especially brought about by the clay fraction, in
particular the sodium-alumio-silicates in the soil.
CEC is an important factor both in agriculture, where it may be related to the binding
capacity of organic compounds and plant nutrients to soil particles, and in sedimentology,
e.g. playing a dominant role in the adsorption characteristics of heavy metals onto lake and
river sediments.

6.5.2 Principle

The experiment is carried out with a percolation tube filled with the soil sample. The soil is
first percolated with NaAc, sodium acetate, to saturate the soil with Na. (All negative places
will be occupied by Na+ ions). After rinsing with ethanol to remove excess of Na+ ions,
another percolation will be carried out with NH4Ac. The adsorbed Na+ will be replaced by
NH4+. The released Na+ is measured by the flame emission spectrophotometry technique.

6.5.3 Apparatus and glassware

a) Atomic absorption spectrophotometer


b) Percolation tube
c) 100 mL and 250 mL volumetric flasks.

6.5.4 Reagents

a) NaAc 1 M; dissolve 34 g NaAc in 250 mL


b) NH4Ac 1 M; dissolve 19 g NH4Ac in 250 mL
c) NH4Ac 0.01 M; dilute 10 mL 1 M NH4Ac to 1 L

d) Ethanol 96%
e) Stock NaCl: dissolve 0.254 g NaCl (dried) in 100 mL. 1 mL=1.00 mg Na
f) Standard serie: dilute 0; 1.00; 3.00; 5.00 and 7.00 mL stock with 0.01 M NH4Ac to
100 mL. This will cover the range of 0-70 mg Na/L

6.5.5 Procedure

1. Weight about 10 g soil with an accuracy of 0.01 g, previously washed with 96% ethanol
and dried at room temperature, add 50 mL inert silver sand and mix
2. Transfer 7 mL inert silversand to the percolation tube which already contains a glass-
wool plug. To ensure a homogeneous packing of the column a special funnel with a
long stem has to be used
3. Transfer in the same way the just prepared soil-sand mixture to the tube
4. Clear the funnel with a brush and add on top of the soil-sand mixture another 7 mL inert
silver sand

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Miscellaneous

5. Percolate with 250 mL 1 M NaAc in about 1 hour (1-2 drops/s) by placing the 250 mL
volumetric flask on top of the tube
6. Percolate with 100 mL ethanol in about ½ hour
7. Discard the effluents
8. Percolate with 100 mL NH4Ac in about ½ hour, collect the percolate in a 100 mL
volumetric flask, fill up with 1 M NH4Ac to the mark and mix

9. Prepare a calibration curve by measuring the emission of the standards at 589 nm


10. Dilute 1.00 mL percolate to 100 mL, measure the emission and determine the Na
concentration

Remark: The soil-sand mixture should always be kept under the liquid level.

6.5.6 Calculation

mg / L ( graph ) x 100
CEC meq Na/100 g = x 100 (dil.factor)
23 x 10 x sample weight ( g )

Literature:
Geochemische bemonsterings- en analysemethoden van Rijkswaterstaat -
Deltadienst. Nota 76-60, bijlage 13.

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6.6 Determination of anion-active detergents

6.6.1 Principle

The cation-active dye methylene blue will form blue complexes with anion-active
components which can be extracted with chloroform. To prevent interferences the extraction
will be carried out from an alkaline solution first, followed by an extraction with the acidic
methylene blue solution.
The absorbance of the organic phase will be measured at 650 nm.

6.6.2 Reagents

a) Buffer solution pH = 10; dissolve 24 g sodiumbicarbonate (NaHCO3) and 27 g water-free


sodium carbonate (Na2CO3) in water and dilute to 1 L
b) Neutral methylene blue solution; dissolve 0.35 g methylene blue in water and dilute to
1000 mL. Prepare this solution at least 24 h. before use. The maximum absorbance of
the blank chloroform phase, measured against chloroform, may not exceed 0.015 for a
1 cm cell at 650 nm
c) Acidic methylene blue solution; dissolve 0.35 methylene blue in water, dilute to 500 mL
and add 6.5 mL conc. H2SO4. Dilute to 1000 mL. Prepare also 24 h. before use. The
absorbance of the blank chloroform phase, measured against chloroform, may also not
exceed a value of 0.015
d) Chloroform, CHCl3 p.a

e) Methyldodecylbenzenesulfonate (= dodecylbenzenesulfonic acid methylester)


f) KOH, 0.1 M solution in ethanol
g) Ethanol, C2H5OH
h) H2SO4, 0.5 M

i) Phenolphthaleine indicator; dissolve 1 g in 50 mL ethanol and add, while stirring,


50 mL H2O. Filter this solution
j) HCl in methanol (CH3OH); mix 250 mL conc. HCl with 750 ml CH3OH

6.6.3 Apparatus and glassware

All glassware should be rinsed with the HCl/CH3OH mixture and washed with H2O

a) Separation funnels of 250 mL


b) Volumetric flasks of 50; 500 and 1000 mL
c) pH meter
d) Spectrophotometer for measurements at 650 nm with 1 and 5 cm cells
e) Filter paper

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Miscellaneous

6.6.4 Procedure

1. Do not sample through a foam layer


2. Filter the sample and discard the first 100 mL of filtrate
3. Transfer a neutralized sample, containing between 20 and 150 μg MBAS, to a 250 mL
separation funnel. Add H2O to a volume of about 100 mL in those cases where the
sample volume is smaller than 100 mL
4. Add 10 mL buffer, 5 mL neutral methylene blue solution and 15 mL chloroform. Mix
vigorously for 1 minute
5. Let the two layers separate and collect the lower (CHCl3) phase in a second separation
funnel which already contains 110 mL H2O and 5 mL acidic methylene blue solution.
Mix vigorously for 1 minute
6. Pass the extract over, previously with ethanol washed and chloroform treated,
glasswool and collect in a 50 mL volumetric flask
7. Repeat step 4, 5 and 6 two times, however, use now 10 mL chloroform
8. Fill up to the mark, with CHCL3, passing over glasswool
9. Measure the absorbance at 650 nm in 1 or 5 cm cells against chloroform.

6.6.5 Calibration

1) Weigh accurately about 425 mg methyldodecylbenzene sulfonate (= dodecylbenzene -


sulfonic acid methylester) and transfer to a round bottom flask
2) Add 50 mL 0.1 M KOH in ethanol and some pumice
3) Reflux for 1 hour
4) Wash the condenser with about 30 mL ethanol and collect this liquid also in the round
bottom flask
5) Neutralize this solution with 0.5 M H2SO4, using phenolphthaleine as indicator
6) Transfer this solution to a volumetric flask of 1000 mL and fill up to the mark with H2O.
Mix the solution. Dilute 25.00 mL to 500.0 mL with H2O

mg weight x 1.023
This standard solution contains μg MBAS per mL
20
7) Add volumes in the range of 0 - 8.00 mL to a 250 mL separation funnel, add H2O to a
volume of about 100 mL and continue as described under “procedure” step 4
8) Prepare a calibration graph by plotting the absorbance against the μg MBAS / 50 mL
endvolume.

6.6.6 Calculation

A sample, treated as described under “procedure”, contains:

μg MBAS in 50 mL endvolume
mg MBAS/L =
mL sample

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6.7 Boron; azomethine-H method

6.7.1 Principle

The water sample is mixed with a buffer masking solution and with Azomethine-H reagent.
After 1-2 hours the absorbance is measured at 410 nm and the concentration can be
calculated by using a calibration graph. The method is fast, has a high sensitivity and
precision compared to other existing spectrophotometric measurements such as the carmine
and the curcumine method. The accuracy of the method is good, recovery is 99-107%. A
detection limit of 0.1 mg B/L is obtained. Common ions of water, wastewater and seawater
do not interfere.

6.7.2 Apparatus

Spectrophotometer for use at 410 nm with 1 cm cells

Reagents

a) Stock 1000 mg/L B solution:


Dissolve 5.716 g H3BO3, boric acid, in 1000 mL H2O and store in a polyethylene bottle.
Do not dry the boric acid at 105 °C or in a dessiccator since it will convert to HBO2,
metaboric acid, due to the loss of water. This HBO2 will not react properly with the
reagents

b) Standard B solutions:
Dilute the stock solutions in order to achieve an intermediate stock solutions of 100 mg/L
B and dilute this intermediate stock solution to achieve a standard serie of 0.0; 0.1; 0.3;
0.5; 0.7; 1.0; 2.0; 3.0; 4.0; 5.0; 6.0 and 7.0 mg/L B.
Keep all standards in polyethylene bottles

c) Buffer masking solution:


Dissolve 250 g CH3COONH4, ammoniumacetate, in 500 mL H2O.
Add 125 mL glacial acetic acid, 6.7 g Na2EDTA and 60 mL thioglycolic acid (80%)

d) Azomethine-H solution:
Dissolve 0.9 g azomethine-H and 2.0 g L(+) ascorbic acid in 100 mL H2O

6.7.4 Calibration and sample treatment

1. Transfer 5.00 mL sample/standard to a plastic vessel, take a smaller volume and add
up to 5.00 mL when the sample concentration exceeds 7 mg/L B

2. Add 4.00 mL buffer masking solution and 2.00 mL azomethine-H reagent

3. Mix well and let react for 1 hour

4. Measure the absorbance of the solutions at 410 nm within 15 minutes

5. Plot absorbance of the standards against the concentration of the standards

6. Measure the absorbance of the sample and use the graph to determine its
concentration, if the sample is diluted one should take the dilution into account

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Miscellaneous

6.7.5 Remarks

1) At UNESCO- IHE some test were done for the removal of boron, samples were filtered
in order to measure the B concentration.
Several filter types were used (e.g. membrane filter OE 67 with a pore size of 0.45 μm
and glass fiber filter GF 52) and checked on the adsorption of BO3-B at different pH
levels.
Also the adsorption of BO3-B onto glass was checked.
The adsorption of BO3-B to filter paper as well to glass was less than 3% at B
concentrations of 0.8 mg/L in the range of pH 3-10

2) Tapwater was spiked BO3-B, the recovery was found to be 103.1%


5x diluted seawater was also spiked and showed a recovery of 100.3%

Literature:
Lopez F.J, Gimenez E and Hernandez F (1993)
Analytical study on the determination of boron in environmental water samples.
Fresenius Journal of Analytical Chemistry, 346: 984-987

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Miscellaneous

6.8 SiO2; molybdosilicate method

6.8.1 Principle

Silica can be present as a monomer, polymer and colloidal silica. Measurement of the
polymer and colloidal forms are difficult and expensive.
Monomeric silica is being measured as the molybdenum reactive silica.
The monomeric or molybdenum reactive silica will react at pH 1.2 with molybdenum complex
to form silicic acids as well as phosphoric acids, which will have a yellow colour. By adding
oxalic acid the phosphoric acids will be destroyed and the silicic acids can be measured at
410 nm by using a spectrophotometer.

6.8.2 Interferences

Coloured compounds , Fe, sulfides and phosphates can interfere.

6.8.3 Apparatus

Spectrophotometer for use at 410 nm with 1 cm cells

6.8.4 Reagents

a) HCl, hydrochloric acid, 6M:


Mix equal volumes of acid with H2O

b) Ammonium molybdate reagent:


Dissolve 15 g (NH4)6Mo7O24 .4H2O in H2O while stirring and gentle heating, dilute to 100
mL. Filter, if necessary.
Adjust the pH to 7-8 with silica free NaOH or NH4OH and store in a polyethylene bottle.
When the pH is not adjusted, a pracipitate will be formed in time. Do not use glass for
storage since silica can be leached from the glass.
Filter the solution again before use, if necessary

c) Oxalic acid:
Dissolve 7.5 g H2C2O4 .H2O in H2O and dilute to 100 mL

d) Monomeric silica standard


Stock solution of 1000 mg/L Si, which is 2139 mg/L SiO2
Certified by Merck, art.nr. 1.02657.0100

6.8.5 Reagent blank purity test

The purity of the reagent water can be tested in the following way:
Transfer to a 50 mL volumetric flask, in rapid succession
- 1 mL solution a)
- 2.00 mL solution b)
- dilute to about 35 mL with H2O and mix
wait 5-10 minutes
- 2.00 mL solution c) and mix
fill up to 50 mL with H2O and mix
Measure the absorbance at 410 nm in a 1 cm cell.
When the absorbance > 0.03 the reagent blank contains too much silica, due to the demi
water used or by contamination of the chemicals. Use in that case Milli-Q water.

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Miscellaneous

6.8.6 Calibration and sample treatment

1) Transfer 0.00; 0.20; 0.25; 0.40; 0.60 and 0.80 mL silica stock solution, or a sample
volume which is less than 40 mL and does not contain more than 1.7 mg SiO2, to 50 mL
volumetric flasks.
The standard serie covers the range of 0-1.711 mg SiO2 / 50 mL

2) Add reagents in the sequence as described under 6.8.5

3) Prepare a calibration graph by plotting the absorbance versus mg SiO2 / 50 mL


volumetric flask

6.8.7 Calculation

1000
SiO2 mg/L = mg SiO2 / 50 mL (from graph) x
V

V = mL sample volume

Literature:
Standard Methods for the Examination of Water and Wastewater, 2o th ed. 1998.
Page 4-156 / 4-158

V.D. Adams, Water and Wastewater Examination Manual, method 4.24 Silica
Molybdosilicate Method, Lewis publishers, 1990, Chelsea, Michigan USA

EPA, Methods for chemical analysis of water and wastes, method 370,U.S. Department of
Commerce, National Technical Information Service (NTIS), march 1993, Cincinatti, OH.

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Miscellaneous

6.9 Selective extraction procedure for As(III)

Extraction performed following Mok et al. (1986), and Mok and Wai (1987) with some
adjustments for IHE laboratory conditions.

Precautions

Here are some points that need some attention before you start this procedure.

a) Before starting this procedure rinse all the glassware with HNO3 to remove any
contamination.
b) Always prepare fresh chemicals (as noted in the chemical listing).
c) Always keep the standards acidified.
d) Before using the separation-funnels, grease the taps well, to prevent leakage.

Reagents

e) Chelating solution, 5% (prepare fresh prior to use)


Dissolve 5.0 grams of NaDDTC in 100 mL demi water.
(Sodium diethyldithiocarbamate (C2H5)2NCSSNa.3H2O;
M=225.31; Baker chemicals).
If necessary (new batch june 2000 does not need to):
Filter the solution over a 0.45-µm membrane filter in order to remove all insoluble
material. Even after filtering the solution might be a little turbid.
Shake with 3-4 mL chloroform per 25 mL NaDDTC to remove bromine and other
impurities from the solution. Now the solution should be clear

f) Extraction solution
Chloroform (p.a); stabilized with 0.75% ethanol. Acros: 23209 –0025

g) Ammonium Citrate buffer, 20%


Dissolve 200 g of di-ammoniumhydrogencitrate in 800 mL demi water.
(M=226.19; quality z.a.; Merck 1.01154.0500)
Adjust the pH to 4.4 with 10M HCl and make up to 1 liter with demi water

h) EDTA solution, 5% (prepare fresh prior to use)


Dissolve 25 gram Na2-EDTA in 500 mL demiwater
(M=372.24; quality p.a. ; Merck 1.08418)

i) HNO3, 50%
Use reagent grade HNO3 (65%), add 500 mL to 150 mL demi

j) HCl (pH adjustment)


Concentrated HCl (32%; 10 M)

k) Stock solution 1000 μg/L As(III)


Prepare a stock solution of 100 mg/L by weighing 132.1 mg dried As2O3 (dried for 2
hours at 105 degrees) to a 1L volumetric flask. Fill up to the mark with 0.01M HNO3.
Prepare a solution of 1000 μg/L (1mg/L) As(III) by transferring 1.00 mL to a 100 mL
volumetric flask and make up the volume with 0.01M HNO3. This solution can be used to
prepare your standards.

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Miscellaneous

Procedure

Extraction of As(III) from water to chloroform

1. Transfer 100 mL of sample to a 250 mL high model bottle with a propylene stopper

2. Add 10 mL ammonium citrate buffer to the sample and mix.


The pH of the solution should for quantitative extraction of As(III) be between 3.5 and
5.5. Adjust with 10M HCl

3. Add 10 mL of a freshly prepared 5% EDTA solution (masking agent) and mix, the
solution should be clear

4. Add 2 mL of a freshly prepared 5% NaDDTC solution and mix.


Tapwater will give a yellow colour (Calcium-complexes)

5. Add 25 mL of chloroform, stopper and extract the mixture by shaking vigorously for 15
minutes on a shaking machine at 300 rpm

6. Transfer the mixture to a separation funnel and allow the phases to separate during 5
minutes. Aqueous phase at top, chloroform phase with As(III) at bottom (Make sure
that no drops of chloroform stay attached to the glass wall or at the surface of the
solution. Slowly swerve the separating funnel and absorb these drops into the solution.
The chloroform drop at the surface can be forced down by pinching it with a metal wire)

7. Rinse and dry the 250 mL bottle and collect the chloroform phase in this bottle, discard
the aqueous phase and wash the funnel with demi water.

B. Back-extraction of As(III) from chloroform to 50 % HNO3

8. Add 5 mL 50% HNO3 solution and shake the mixture for 10 minutes vigorously by
shaking at 300 rpm., followed by manual shaking during 1 minute

9. Transfer the mixture to the separation funnel, let the phases separate for 5 minutes.
Discard the lower, chloroform, phase (25 mL)
(Make sure that no drops of chloroform stay attached to the glass wall or at the surface
of the solution. Slowly swerve the separating funnel and absorb these drops into the
solution. The chloroform drop at the surface can be forced down by pinching it with a
metal wire)

10. Collect the acid phase in a 100 mL volumetric flask.


Rinse the funnel a few times with demi water. Fill the volumetric flask to the mark with
demiwater.

Measurements of As on GF-AAS

Expected As(III)< 60 μg/L :


Add 1 mL matrix-modifier ( i.e. 25% Ni(NO3)2 ) on top of the 100 mL for analysis with the
Graphite Furnace AAS. Detection limit is 4 ug/L.

Expected As(III)> 60 μg/L :


Dilute a certain known volume to 100.0 mL and add 1 mL of matrix modifier.
(i.e. 25% Ni(NO3)2 ) on top of the 100 mL for analysis with the Graphite Furnace AAS.

*Remark: It is also possible to add the matrix modifier (MM) in proportion to the amount of
sample upon analysis. (ex. use 20 μL MM on top of 2.00 mL sample).

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Miscellaneous

6.10 Colorimetric Method for determination of Chromium (VI)

6.10.1 Principle

This procedure measures only hexavalent chromium (Cr6+). Therefore, to determine TOTAL
chromium convert all the chromium to the hexavalent state by oxidation with potasssium
permanganate after digesting with a sulfuric-nitric acid mixture. The hexavalent chromium is
determined colorimetrically by reaction with diphenylcarbazide in acid solution. A red-violet
color of unknown composition is produced and is nearly specific for chromium.

6.10.2 Interferences

1. Mo and Hg (hexavalent) at concentrations higher than 200 mg/L


2. Va at concentrations 10 times higher than Cr.
3. Fe at higher concentrations because of orange color starting from 1 mg/L.

To remove the above interferences, refer to the procedure in standard methods

6.10.3 Apparatus

1. Spectrophotometer for use at 540 nm with 1 cm cells.

6.10.4 Reagents

1. Stock of chromium solution:


Dissolve 141.4 mg K2Cr2O7 in water and dilute to 100 mL (1.00 mL=500 ug Cr)
2. Standard chromium solution:
Dilute 1.00 mL stock chromium solution to 100 mL (1.00 mL = 5 μg Cr)
3. Diphenylcarbazide solution:
Dissolve 250 mg 1,5-diphenylcarbazide in 50 mL acetone. Store in a brown bottle and
discard the solution when it becomes discolored.
4. Sulfuric acid, H2SO4, conc.
5. Sulfuric acid, H2SO4, 1+1.
6. Sulfuric acid, H2SO4 0.2 M:
Dilute 5.5 mL of H2SO4, conc. to 500 mL with demineralized water.
7. Ammonium hydroxide, NH4OH, conc.
8. Phosphoric acid, H3PO4, conc.
9. Methyl orange indicator solution.
10. Potassium permanganate solution:
Dissolve 4 g KMnO4 in 100 mL demineralized water.
11. Sodium azide solution:
Dissolve 0.5 g NaN3 in 100 mL water.

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Miscellaneous

6.10.5 Calibration

Remark: The volumes mentioned in Standard Methods are for 100.00mL volumetric flasks,
here the volumes are decreased by a factor 2 to limit the waste.
1. Transfer the following amounts of standard Cr(VI) to 50.00 mL volumetric flasks:
0; 5.00; 10.00; 15.00; 20.00; 25.00 and 30.00 mL.

2. If necessary, adjust the pH to 1.0 ± 0.3 with 0.2 M H2SO4.


3. Fill up to the mark with demineralized water and mix
4. Add 1.0 mL diphenylcarbazide solution and mix.
5. Let stand for 5 to 10 min for full color development
6. Transfer an appropiate portion to a 1-cm absorption cell and measure its absorbance at
at 540 nm. Use demineralized water as reference.

6.10.6 Procedure for Cr(VI) 0.1 – 3 mg/L Cr(VI)/L

1. Transfer an appropiate amount of the (filtered) sample to a volumetric flask of 50.00 mL.
2. Adjust the pH to 1.0 ± 0.3 with 0.2 M H2SO4.
3. Fill up to the mark with demineralized water and mix
4. Add 1.0 mL diphenylcarbazide solution and mix
5. Let stand for 5 to 10 minutes for full color development
6. Transfer an appropiate portion to a 1-cm absorption cell and measure its absorbance at
at 540 nm. Use demineralized water as reference.

6.10.7 Cr total 0.1 – 1 mg/L

To compensate for possible slight losses of chromium during digestion or other analytical
operations, treat chromium standards by the same procedure as the sample.

Procedure for Cr total

1. Pipet measured volumes of sample or standard chromium solution (5 μg/mL) ranging


from 1.00 to 10.00 mL, to give standards of 5 to 50 μg Cr, into a 250 mL conical flask.
2. Add several drops of methyl orange indicator, then add conc. NH4OH until solution just
begins to turn yellow.
3. Add 1+1 H2SO4 dropwise until it is acidic, plus 1 mL (20 drops) in excess.
4. Adjust volume to about 40 mL, add a boiling chip, and heat to boiling.
5. Add 2 drops KMnO4 solution to give a dark red color. If fading colors, add KMNO4
dropwise to maintain a excess of 2 drops.
6. Boil for 2 more minutes.
7. Add 1 mL NaN3 solution and and continue boiling for approxiately 30 seconds, add
another 1 mL NaN3 solution.
8. Continue boiling for 1 min after color has faded completely, then cool.
9. Add 0.25 mL (5 drops) H3PO4

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Miscellaneous

10.Continue from 6.10.6.1 [endvolume after digestion must be lower than 50 mL to


continue with 50 mL volumetric flasks]

6.10.8 Calculations

[Cr] mg/L = (μg Cr in 51 mL endvolume / mL original sample) * 1000

For digested samples:

[Cr] mg/L = (μg Cr in 51 mL endvolume / ( A * B )) * 1000

where: A = mL original sample, and


B = mL portion from 50 mL digested sample

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Miscellaneous

6.11 Determination of CaCO3 in sediment

6.11.1 Introduction

Depending upon climate and local conditions, carbonates may be the dominant sink for
some trace elements. The major control on trace element uptake by carbonates, often in
metastable and and polymorphic forms, is pH. Trace elements may be coprecipitates as
carbonates or, like Cd, may actually replace Ca2+ in the lattice.
The preferred method of selective dissolution of the carbonate phase is an acidified acetate-
buffered reaction, namely 1 M NaOAc/HOAc at pH 5. Grosssman and Millet (1961) reported
that organic carbon and free iron concentrations in noncalcareous soil samples were
unchanged after contact with this buffer for nine weeks; other workers (Nissenbaum, 1972,
Gupta and Chen, 1975 and McLaren and Crawford, 1973) have demonstrated that lower pH
values lead to a partial attack of Fe and Mn oxides. The time required for carbonate
dissolution will depend upon such factors as particle size, percentage and type of carbonate
present, and sample size. Tessier et al. (1979) evaluated the optimum time for leaching the
carbonate fraction and their results indicated complete Ca dissolution within 5 hr of leaching.
This extraction time is likely to be sufficient for most finely devided suspended solids.
However, for coarse bottom sediments with high carbonate content, longer leaching times
and frequent pH adjustment might be necessary.

6.11.2 Procedure for 1 gram sediment (dry weight)

1. In order to remove the exchangeable Ca fraction, the sediment is extracted at room


temperature for 1 hr with 8 mL 1 M MgCl2 solution with continuous agitation.
2. The residue is leached at room temperature for at least 5 hr with 8 mL of 1 M NaOAc
adjusted to pH 5 with acetic acid (HOAc) with continuous agitation.
3. Determine the Ca-concentration in centrifuged supernatants.

Remark: Leaching the exchangeable fraction is frequently carried out with NaOAc rather than MgCl2.
This may lead to the formation of a complex Ca2+ + Oac- = CaOAc+ (log K = 1.24), which might reduce
the Ca-yield after step 2. Therefore, MgCl2 is to be preferred.

References
Grossman, R.B. and J.C. Millet, 1961. Soil Sci. Soc. Am. Proc., 25, 325-326
Gupta, S.K. and K.Y. Chen, 1975. Environ. Lett., 10, 129-158.
McLaren, R.G. and D.V. Crawford, 1973. J. Soil Sci., 24, 172-181
Nissenbaum, A., 1972. Isr. J. Earth Sci., 21, 143-154
Tessier, A., P.G.C. Campbell, and M. Bisson, 1979. Anal. Chem. Vol. 51, No. 7, 844-851.

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Miscellaneous

6.12 Acid Volatile Sulphide, AVS, in sediment


Colorimetric Method

6.12.1 Introduction

Acid volatile sulphide (AVS) is a measure for the available amount of sulphide in the
sediment.
It is also known as the fraction of sulphides extracted by cold hydrochloric acid.
AVS consists primarily of iron mono sulfides and manganese monosulphides.
AVS can bind metals in anaerobic sediments and thereby reduce or eliminate the toxicity of
the metals. Therefore AVS is a key parameter to control the behavior of some metals in
sediments.

The colorimetric method substitutes the gravimetric method. The gravimetric method yields a
low recovery and is time consuming compared to the colorimetric method.

6.12.2 Materials

• 1 reaction vessel with 3 openings (three-necked round bottom flask)


• 1 or 2 gas-wash bottles (trapping vessels)
• Magnetic stirrer
• Tubing for N2 flushing
• Stoppers
• Clamps

6.12.3 Chemicals

a) 6M HCl:
Dilute 60mL of concentrated HCl to 100mL

b) 1M H2SO4:
Dilute 55mL of concentrated H2SO4 to 1L (Add the acid to the water !)

c) 0.5M NaOH:
Dissolve 20 g NaOH in 1L.

d) Mixed Diamine Reagent (MDR):


MDR consists of two mixtures.

Solution A:
add 330mL concentrated H2SO4 to 170mL of demiwater.
After cooling to room temperature add 1.125g of
N,N-dimethyl-p-phenylenediamine oxalate and dissolve.
(NOTE: N,N-dimethyl-p-phenylenediamine oxalate is very toxic !!)

Solution B:
dissolve 2.7g FeCl3.6H2O in a mixture of 50 mL conc. HCl + 50mL H2O

Mix solutions A and B in a ratio of 5 : 1

e) Stock Na2S solution:


Dissolve 6 g of 35% Na2S (this is equal to about 2 g of Na2S) in 250mL 0.1M NaOH. That
is a 0.1M S2- solution.
Standardize this solution with an iodine solution of a known concentration in order to

99
Miscellaneous

know the exact concentration of S (see Appendix)

f) Standard Na2S solution, 500 µmol S2-/ L:


Dilute the stock solution about 200x, depending on the concentration of the stock
solution, with 0.1M NaOH to get a 500 µmol S2-/L working solution.

6.12.4 Procedure sediment in reaction vessel

1. Prepare a setup consisting of a reaction vessel and one or two trapping vessels
(depending on amount of S2- expected).
The trapping vessels contain 80mL of 0.5M NaOH.

2. Weigh accurately about 0.5 to 1.0 g of wet sediment and add 50 mL of demiwater to the
reaction vessel.
(Weigh more if S2- is expected to be < 0.25 µmol/g)

3. Stir the suspension for 10 minutes while flushing with N2-gas.

4. Close the N2 gas.

5. Add 10 mL of 6M HCl to the reaction vessel.


(Final concentration of solution is 1M HCl)

6. Start the N2 gas.

7. Continue for 1 hour while stirring and flushing with N2-gas at room temperature.
The evolved H2S is trapped in the trapping vessels (NaOH).

8. The sulfide concentration in the trapping vessels is determined with a spectrophotometer


at 670nm in a 1cm cuvet (according to Kloster and King 1990).

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Miscellaneous

6.13 Spectrophotometric determination of S2- in the sample

The reagent used to determine the sulphide concentration in the trapping vessels is called
Mixed Diamine Reagent (MDR, see 1.3C). The N,N-dimethyl-p-phenylenediamine oxalate in
the MDR reacts with iron(III)sulphide and hydrogensulphide in a strong acid environment.
The result is a methylene blue color.

6.13.1 Calibration

Range: 0 – 2.5 µmol S2-/ 100 mL


1. Add 0, 1, 2, 4 and 5mL of standard solution to 100 mL volumetric flasks. (Respectively:
0, 0.5, 1.0, 2.0 and 2.5 µmol S2- )
2. Add 80mL of 0.5M NaOH to each flask
3. Add 10mL of MDR
4. Fill up to the mark with demiwater
5. Measure the absorbance after 30 minutes at 670 nm in a 1cm cuvet
6. Plot absorbance versus µmol S2-/100 mL

6.13.2 Measurement of S2- in the sample, concentration < 2.5 µmol S2-/g

1. Transfer the content of the reaction vessel into a 100 mL volumetric flask
2. Rinse the reaction vessel
3. Add 10mL of MDR to the reaction vessel
4. Fill up to the mark with demiwater
5. Measure the absorbance after 30 minutes at 670 nm in a 1cm cuvet
6. Determine µmol S2-/100 mL

6.13.3 Measurement of S2- in the sample, concentration > 2.5 µmol S2-/g (see remarks!)

1. Transfer the content of the reaction vessel into a 100 mL volumetric flask
2. Rinse the reaction vessel
3. Fill up to the mark with demiwater
4. Transfer a portion (A) into a 100 mL volumetric flask
5. Add 0.5M NaOH till ca. 80mL
6. Add 10mL of MDR
7. Fill up the mark with demiwater
8. Measure the absorbance after 30 minutes at 670 nm in a 1cm cuvet
9. Determine µmol S2-/100 mL

6.13.4 Calculations

Concentration < 2.5 µmol S2-/g

umol S / 100 mL
µmol S2-/g =
weight ( g ) * dryweight content ( g / g )

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Miscellaneous

6.13.5 Remarks

1. N,N-dimethyl-p-phenylenediamine oxalate is a toxic compound take proper precautions.


2. The recovery % for this procedure is over 90%
3. The Spectrophotometric procedure for high concentrations has not been tested as
written in this procedure. According to the official procedure the sample is diluted with
1M H2SO4 after color development. This has to be tested before it can be applied..

Literature:

Van den Hoop et. al., chemosphere, vol 35, no10, pp 2307-2316, 1997

Allen et al., Environmental toxicology and chemistry, vol 12, pp 1441-1453, 1993
Kloster and King, Journal American water works association, vol 69, no10, pp 544-546,
1977, The determination of sulfide with DPD

Standard Methods 18th edition page 3-59

102
Appendix 1

APPENDIX 1

STANDARD SUBSTANCES AND SOLUTIONS; FILTER PAPER

A. Some primary standards and their treatment

With the exception of gravimetric procedures, all laboratory-born results are based on
comparison with a standard. So, a correct standard is of utmost importance for reliable
analytical results. There are two categories of standards: primary and secondary.

A primary standard should have the following characteristics:


- available in a pure state
- be unaltered during weighing and storage
- have high molecular mass
- be readily soluble
- react instantaneously and completely

Some substances used for primary standards are listed in the table below. Each of these has to
be dried at optimum temperature just before weighing, according to this (incomplete) table:

Primary standard Recommended pretreatment


Temperature Time (h)
(ºC)
Calcium carbonate 105 2
Sodium carbonate 270 ± 10 2
Sodium chloride ≈ 200 24
Sodium oxalate 105 2
Potassium hydrogen 105 2
phthalate
Potassium iodate 180
Arsenic trioxide (poison!) 105 - 110
Potassium dichromate 150 - 200 3

Just before drying, any lumps should be cut down so that only fine crystals remain. This is
important, because coarse crystals may contain occluded water in their cavities and thus would
need a higher drying temperature. The fine crystals should, however, not be pulverized because
this enhances deliquescence (= attraction of water) and/or efflorescence (= weathering) later
on. Besides, the heating oven should be ventilated well, since this decreases both the time and
the temperature of drying. After the recommended heating procedure, the primary standard
substance should be cooled in a desiccator containing preferably magnesium perchlorate as a
dessicant. The dried substance should be weighed as soon as it has reached the ambient
temperature.
Primary standard substances should be stored in closed weighing bottles which are placed in a
desiccator. (Note: this only holds for substances not containing crystal water, and for
substances that can be dried). The desiccator may contain a desiccant like silicagel (which
should be blue). In this way, a primary standard may be stored for weeks or months without
appreciable uptake of water. It is advisable, however to repeat the drying procedure now and
then.
As a rule, hydrated substances are not suitable for use as a primary standard, because of
difficulties in keeping the amount of crystal water constant.
Some substances are not allowed to be dried, even though they do not contain crystal water.
This holds e.g. for boric acid, H3BO3, because drying would convert it to metaboric acid, HBO2.

103
Appendix 1

104
Appendix 1

B. Secondary standard substances

A secondary standard is substance which may be used for standardizations after having been
compared against some primary standard. Many hydrated reagents fall into this category. Two
of them are of practical importance: oxalic acid and borax.
Oxalic acid, (COOH)2.2H2O, may be stored over a saturated solution of NaBr. It may be more
convenient, however, to check the content by titrating with standardized KMnO4. In either case
it seems wise to purchase analytical grade oxalic acid.
Borax, Na2B4O7.10H2O, has the advantage of a high molecular mass in comparison to oxalic
acid. Borax may be stored over a saturated solution of NaCl or NaBr. For example, a sample of
borax decahydrate (stored in the bottle in which it had been supplied) can loose 25% of its
crystal water in a period of 2 years! They recrystallize borax instead of storing it over a
deliquescing salt.
Hydrated substances should never be pulverized, because this accelerates the process of
efflorescence greatly.

C. Preparation of standard solutions

Reagents

The purity of reagents should be chosen in accordance with the intended applications. It is
common practice to use the “analytical reagent” quality, which contains contaminations at
mg/kg level. For routine analysis of macro elements, however, the “chemically pure” grade
might suffice. For the determination of micro elements, on the other hand, it might be necessary
to use high-purity chemicals, which are specified for different techniques (“spectroscopically
pure”, “chromatographically pure” ). Since such reagents are very expensive, the need for their
application should be established first.

Water

The water used for laboratory work has to meet several requirements concerning its purity,
among which pH and conductivity are the main ones. The pH may vary between 5.0 and 6.5,
while the electrical conductivity should be lower than 1 μS cm-1.
Two ways of purification are well-known and practised generally: distillation and deionisation
(the latter is also called demineralisation). Each technique is appropriate for normal analytical
work. However, after single distillation (from hard glass apparatus) traces of metal ions are still
present; deionisation, on the other hand, may leave traces or organic substances in the water.
For special purposes, e.g. determination of very low contents of trace elements, the two
techniques may be applied both, thus producing “demi-dest” water. In some cases, e.g. for the
titrimetric determination of low levels of carbonate, it is necessary to use CO2-free water. This is
simply obtained by boiling for 4 minutes; during cooling the vessel should be stoppered by a
guard tube containing a CO2 absorbent, but in practice a watch glass has proved to be
sufficient.

Solutions

All standard substances are only applied in solution, and are accordingly called primary
standard solutions and secondary standard solutions. The preparation of such solutions
(preferably in 0.1 M acid) is usually straightforward: weighing, dissolving and making up the
volume. Of course, the substance must be weighed very precisely and prepared in a volumetric
flask. In this way, solutions of rather high concentrations (usually 1000 mg element per litre) are
prepared; these are called stock solutions. For several substances there are stock solutions
commercially available in sealed ampoules. This may be convenient, e.g. when the standard
substances does not dissolve readily.

105
Appendix 1

106
Appendix 1

Stock solutions are preferably stored cool and dark, either in hard glass or in polythene vessels,
flushed in advance with acid, and may thus be stable for months. When stock solutions are
stored for more than a year, they should be checked before using; it is known, for example, that
storage in a polythene flask may lead to 1 - 1.5% loss of water by evaporation.
From those stock solutions all other solutions are made by dilution, these are called standard
solutions. Standard solutions may deteriorate rather rapidly due to adsorption. It is advisable to
check the rate of decomposition, and to prepare fresh standard solutions at an appropriate
frequency, if necessary every day.

D. Filter paper

Filter paper is classified from coarse to fine according to its porosity. The following table gives
this classification for some well-known brands of quantitative papers, i.e. HCl-HF treated papers
(formerly called “ash-free”).

Macherey Schleicher Whatman hardened*


Porosity
& Nagel & Schüll )
quantitative paper
very fine MN 640 dc S/S 589/3 42 542
fine MN 640 d S/S 589/5
medium fine MN 640 m S/S 589/2 40 540
coarse MN 640 w S/S 589/1 41 541
phase separating
paper
medium MN 616 WA S/S 597 hy 1PS

*) especially for filtration with Büchner funnels

Glass fiber filters:

Schleicher Whatman
Efficiency
& Schüll
98% for > 1.6 μm GF/A
98% for > 1.0 μm GF/B*
98% for > 1.2 μm GF52 GF/C
98% for > 2.7 μm GF/D
98% for > 0.7 μm GF/F*
99.9% for > 0.3 μm* GF6*

* For chlorophyll determination

Literature

T. Yoshimori. Drying and weighing of standard reference materials for titrimetric analysis and
the status of the Faraday constant as an international standard. Talanta 22 (1975) 827-825.

107
Appendix 2

APPENDIX 2

STANDARDIZATION OF 0.1 M HCl OR 0.05 M H2SO4 WITH Na2CO3

Principle

The acid is standardized by titration of sodium carbonate with methylred-bromocresol green as an


indicator.

Reagents

Sodium carbonate. Na2CO3, pretreated according to Appendix 1


Methylred-bromocresol green: a) Suspend 0.15 g bromocresol green in 100 mL 96% ethanol,
add dropwise 0.1 M NaOH until the red colour turns into dark
red (about 5 mL) dissolve by stirring. If the indicator is available
as its sodium salt, it is sufficient to dissolve 0.15 g in 100 mL
96% ethanol.
b) Dissolve 0.1 g methylred in 100 mL 96% ethanol.
c) Mix equal volumes of a) and b).

Procedure

Weigh out accurately about 200 mg of sodium carbonate and transfer it to a 300 mL erlenmeyer flask.
Dissolve in about 75 mL of distilled water and add 3 drops of the indicator. Titrate with the acid until
the colour has changed from green via grey to rose. Then boil until the CO2 is driven out of the
solution, cool and titrate further until the colour has become rose again.

Calculation

The molarity of the acid is:

w
M = 0.01887 * for HCl
V
w
or M = 0.009435 * for H2SO4
V
in which:

w = weight of sodium carbonate, in mg;


V = volume of acid added, in mL.

Remarks

1. The colour change before the boiling stage should be reached 0.2 - 0.3 mL before the
equivalence point. After boiling, the consumption of acid should not exceed 0.3 mL, otherwise
the determination should be repeated.
2. Dried sodium carbonate is very hygroscopic.

108
Appendix 3

APPENDIX 3

STANDARDIZATION OF 0.1 M NaOH OR KOH WITH OXALIC ACID

Principle

The base is standardized by titration of oxalic acid with methyl red as an indicator.

Reagents

Oxalic acid. (COOH)2.2H2O, pretreated according to Appendix 1.


Calcium chloride. Dissolve 200 g of CaCl2.6H2O in 1 liter of water; neutralize with respect to methyl
red.
Methylred, dissolve 0.1 g in 100 mL 96% ethanol.

Procedure

Weigh out accurately 250 mg of oxalic acid and transfer it to a 300 mL erlenmeyer flask. Dissolve the
acid in about 100 mL of distilled water (free from CO2), add 3 drops of indicator solution and titrate
with the base until the colour of the indicator has changed to yellow. Then add 10 mL of the calcium
chloride solution and proceed with the titration until the colour has once again changed to yellow.

Calculation

The molarity of the base is:

w
M = 0.01586 *
V
in which:

w = weight of oxalic acid, in mg


V = volume of base, in mL.

Remarks

1. Soldium oxalate reacts alkaline with respect to the indicator. Therefore, calcium chloride is
added which results in a precipitation of calcium oxalate and the consequent liberation of HCl;
this HCl is neutralized in the final stage of titration.

2. To prevent coprecipitation of oxalic acid, the solution of calcium chloride is added at the end of
the titration.

3. Small amounts (200 - 500 mL) of CO2 free water can be prepared by boiling during 4 minutes in
erlenmeyer flasks (not in beakers). It is allowable to cool while only a watch glass covers the
erlenmeyer flask.

109
Appendix 4

APPENDIX 4

STANDARDIZATION OF 0.02 M KMnO4 WITH SODIUM OXALATE

Principle

A KMnO4 solution is standardized by oxidimetric titration of sodium oxalate.

Reagents

Sodium oxalate (COONa)2, pretreated according to Appendix 1.


Sulphuric acid, H2SO4 2 mol/L. Add carefully, while swirling, 56 mL of concentrated sulphuric acid
(96%) to about 400 mL water. Allow to cool and make up to 500 mL.
Potassium permanganate solution, KMnO4 0.02 mol/L. Dissolve 3.2 g KMnO4 in about 800 mL of hot
distilled water and then boil for about 5 min. Cool, filter over a 0.45 μm membrane filter or a very fine
glass frit and make up to 1 liter with water.

Procedure

Weigh out accurately about 275 mg of sodium oxalate and transfer it to a beaker of 600 mL. Dissolve
the substance, while stirring, in a mixture of 100 mL of 2 M sulphuric acid and 100 mL of water at
about 27 ºC.
While stirring, add with a burette about 35 mL of the permanganate solution and wait until the solution
has become colourless. Heat to 55-65ºC and then titrate dropwise, while stirring, until a rose colour
persists after a waiting period of 30 seconds.
Perform a blank determination with a mixture of 100 mL of 2 M sulphuric acid and about 140 mL of
water.

Calculation

The molarity of the permanganate solution is:

w
M = 0.002987 *
a −b

in which

w = weight of the sodium oxalate, in mg;


a = volume of KMnO4 added to the analyte solution, in mL;
b = volume of KMnO4 added to the blank solution, in mL;

110
Appendix 5

APPENDIX 5

STANDARDIZATION OF 0.1 M AgNO3 WITH NaCl (according to Mohr)

Principle

A known amount of NaCl is titrated with an AgNO3 solution, so that silver chloride precipitates. After
the equivalence point, any excess of AgNO3 forms a red precipitate of silver chromate with the
indicator.

Reagents

Silver nitrate solution, AgNO3 0.1 mol/L. Dissolve 17.0 g of AgNO3 in 1 liter of water.
Potassium chromate solution. Dissolve 5 g of K2CrO4 in 100 mL of water.
Sodium chloride. NaCl, pretreated according to Appendix 1.
Calcium carbonate, CaCO3 (analytical grade).

Procedure

First perform a blank determination in a 250 mL beaker that contains 140 mL of water, 4 mL of
potassium chromate solution and 0.5 g of CaCO3. Titrate with the silver nitrate solution until the
suspension shows a weak, but distinct, red colour which persists even with energetic stirring. Keep
this suspension for future comparison.
Then weigh out accurately about 250 mg of sodium chloride, transfer it to a 250 mL beaker, and add
100 mL of water and 4 mL of potassium chromate solution. Titrate carefully, while stirring, until the red
colour which appears with every drop of AgNO3 fades aways slowly. Then titrate dropwise until the
solution shows the same shade of red as the blank.

Calculation

The molarity of the silver nitrate solution is:

w
M = 0.01711 *
a −b

in which

w = weight of NaCl, in mg;


a = volume of AgNO3 added to the analyte solution, in mL;
b = volume of AgNO3 added to the blank solution, in mL;

111
Appendix 6

APPENDIX 6

STANDARDIZATION OF 0.05 M EDTA WITH CaCO3

Principle

An EDTA solution is standardized by titration of CaCO3 at pH 10 with Eriochrome Black T as an


indicator.

Reagents

HCl 1 M. Add 8.3 mL of concentrated hydrochloric acid (36%) to about 40 mL water and make up to
100 mL.
Buffer solution, pH 10. Dissolve 5.4 g of ammonium chloride, NH4Cl, and 0.2 g of Mg EDTA, in 35 mL
of concentrated aqueous ammonia (25%) and dilute with water to 100 mL.
EDTA solution, 0.05 mol/L. Dissolve 18.6 g of disodium ethylenediamine tetra acetate,
Na2EDTA.2H2O, in 1 liter of water.
Eriochrome Black T, dissolve 0.5 g in 90 mL triethanolamine.

Procedure

Weigh out accurately about 150 mg of CaCO3 into a 300 mL erlenmeyer flask. Dissolve it in a small
excess of 1 M HCl (about 3 mL). Dilute to about 100 mL and boil for some minutes. Check whether all
CaCO3 has dissolved; if not, add 0.5 mL of 1 M HCl extra and boil again. Then add to the still hot
liquid 10 mL of the buffer solution and 0.4 mL of the indicator solution. Titrate from red to a blue end
point with the EDTA solution.
Perform also a blank determination.

Calculation

The molarity of the EDTA solution is:

w
M = 0.009991 *
a −b

in which
w = weight of CaCO3, in mg;
a = volume of EDTA used for the analyte, in mL;
b = volume of EDTA used for the blank, in mL;

Remarks

1. If, by accident, too much HCl has been added, the solution can be neutralized first with 1M
ammonia.
2. Store the standardized EDTA solution in polythene bottles.
3. The Na2EDTA should be of analytical grade, since lower grades may contain other complexing
agents which can give erroneous results (e.g. in the determination of Zn).
4. The addition of Mg EDTA to the buffer solution provides a sharper colour change at the end
point when using Eriochrome Black T.

112
Appendix 7

APPENDIX 7

STANDARDIZATION OF 0.025 M Na2S2O3 WITH POTASSIUMDICHROMATE

1. To 80 mL distilled water in an erlenmeyer flask add 10 mL 10% KI solution and 10 mL 4 M HCl.


2. Add 20.00 mL of a 1.2259 g/L K2Cr2O7 solution (Dried at 103ºC before weighing).
This K2Cr2O7 solution is equal to a 10 times diluted 0.0417 M solution which is also used for
COD determinations.
3. Let reaction take place for 6 min. in the dark.
4. Titrate with Na2S2O3, at the end of the titration 2 mL 0.5% starch solution must be added.

Calculation

0 .500
The molarity of the Na2S2O3 solution =
mL Na 2 S2 O3

113
Appendix 8

APPENDIX 8

Standardization of 0.025 N I2 solution with 0.025 N Na2S2O3.

Chemicals:

• Starch 5%: dissolve 5 g of starch in 100mL hot demi water.


• Sodium thiosulfate solution, 0.025N Na2S2O3
6.2g sodium tiosulfate in 1000mL.
(Standardize according to appendix 7 in the lab manual)
• Iodine solution, 0.025N:
Dissolve 20 to 25 g KI in a little water and add 3.2 g iodine. After the iodine has dissolved,
dilute to 1000mL.

Procedure:

1) Pipet 25.00 mL of 0.025 N thiosulfate into an Erlenmeyer flask


2) Add 5 drops of 5% starch
3) Titrate with 0.025 N iodine until the color turns blue
4) Calculate the concentration of the iodine solution

Calculations:

N thiosulfate * 25 mL
Normality of Iodine =
mL iodine

114
Appendix 9

Appendix 9

Standardization of stock 0.1M Na2S solution with I2 solution.

Chemicals:
• 6M HCL
• Starch 5% : dissolve 5 g of starch in 100mL hot demi water.
• Standard iodine solution, 0.025 N:
Dissolve 20 to 25 g KI in a little water and add 3.2 g iodine. After the iodine has dissolved,
dilute to 1000mL
• Sodium thiosulfate solution, 0.025N Na2S2O3
6.2g sodium tiosulfate in 1000mL.
(Standardize against K2Cr2O7 according to appendix 7 in the Lab manual)

Procedure:
1) Measure from a buret an amount of I2 solution estimated to be an excess over the
amount of S2- present.
2) Add distilled water, if necessary, to bring volume to about 20mL
3) Add 2 mL 6M HCL
4) Pipet 200 mL sample into the flask, discharging sample below the solution surface
5) If the I2 color disappears, add more I2 solution until color remains
6) Back-titrate with 0.025N Na2S2O3, adding a few drops of starch solution as you
approach the end point, continue until blue color (of starch) disappears.

Calculations:
1.00 mL of 0.0250N I2 solution reacts with 0.4 mg S2-

[( A * B) − (C * D)] *16000
mg S2-/L =
sample volume(mL)
Where:
A) mL iodine
B) normality of iodine
C) mL Na2S2O3
D) normality of Na2S2O3

115
Appendix 10

APPENDIX 10

ATOMIC WEIGHTS

The atomic weights used throughout these appendices are the standard atomic weights 1987 as
given by the IUPAC. The following table is a selection of the existing elements. The values are given
with the same number of decimals as in the original IUPAC table, i.e. with the same degree of
confidence. The last column gives the uncertainty of the last decimal digit. The values apply to
elements as they exist naturally on earth, and are thus applicable to any normal material.

Name Symbol Atomic nummer Atomic weight Last decimal


silver Ag 47 107.8682 ±2
aluminium Al 13 26.981539 ±5
arsenic As 33 74.92159 ±2
boron B 5 10.811 ±5
barium Ba 56 137.327 ±7
bismuth Bi 83 208.98037 ±3
bromine Br 35 79.904 ±1
carbon C 6 12.011 ±1
calcium Ca 20 40.078 ±4
cadmium Cd 48 112.411 ±8
cerium Ce 58 140.115 ±4
chlorine Cl 17 35.4527 ±9
cobalt Co 27 58.93320 ±1
chromium Cr 24 51.9961 ±6
cesium Cs 55 132.90543 ±5
copper Cu 29 63.546 ±3
fluorine F 9 18.9984032 ±9
iron Fe 26 55.847 ±3
hydrogen H 1 1.00794 ±7
mercury Hg 80 200.59 ±3
iodine I 53 126.90447 ±3
potassium K 19 39.0983 ±1
lanthanum La 57 138.9055 ±2
lithium Li 3 6.941 ±2
magnesium Mg 12 24.3050 ±6
manganese Mn 25 54.93805 ±1
molybdenum Mo 42 95.94 ±1
nitrogen N 7 14.00674 ±7
sodium Na 11 22.989768 ±6
nickel Ni 28 58.69 ±1
oxygen O 8 15.9994 ±3
phosphorus P 15 30.973762 ±4
lead Pb 82 207.2 ±1
sulphur S 16 32.066 ±6
antimony Sb 51 121.75 ±3
selenium Se 34 78.96 ±3
silicon Si 14 28.0855 ±3
tin Sn 50 118.710 ±7
strontium Sr 38 87.62 ±1
titanium Ti 22 47.88 ±3
zinc Zn 30 65.39 ±2

Literature

116
Appendix 9

IUPAC commission on Atomic Weights and Isotopic Abundances: Atomic Weights of the Elements
1987. Pure Appl. Chem. 60 (1988) 841-854.

117