Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis

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A PRACTICAL GUIDE TO THE SAMPLE COLLECTION,
PRESERVATION AND ANALYSIS

College of Aquaculture and Fisheries

Prepared by:

HUYNH TRUONG GIANG, Ph.D.

Water Quality Study Lab
Faculty of Aquatic Biology and Environmental Science

Can Tho University

 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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COLLECTION AND PRESERVATION OF SAMPLES

1. Type of sampling:
Two different types of sample can be taken from rivers, lakes and similar surface waters
including composite or integrated samples. Composite samples may be of the following types:
 Depth-integrated: most commonly made up of two or more equal parts collected at
predetermined depth intervals between the surface and the bottom.
 Area-integrated: made by combining a series of samples taken at various sampling
points spatially distributed in the water body. This type usually is used to collect the
sample in shrimp ponds, catfish ponds...
 Time-integrated: made by mixing equal volumes of water collected at a sampling
station at regular time intervals.
 Discharge-integrated: It is first necessary to collect samples and to measure the rate
of discharge at regular intervals over the period of interest. A common arrangement is
to sample every 2 hours over a 24-hour period. The composite sample is then made by
mixing portions of the individual sample that are proportional to the rate of discharge
at the time the sample was taken.
2. Sample containers:
Container Min. sample Preservation Maximum
Determination size (mL) storage
Analyze in field
Temperature
immediately
Analyze in field
pH
immediately
Analyze in field
Transparence
immediately
Analyze in field
Conductivity
immediately
Analyze in field
Salinity
immediately
Turbidity P 100 Refrigerate Analyze same day
Add 1mL MnSO4
DO G 125 and 1 mL KI- 2-3 days
NaOH
Alkalinity P 200 Refrigerate 14 days
Hardness P 200 Refrigerate 14 days
COD G 125 Add 2mL H2SO44M 7 days
TSS P 200 Refrigerate Analyze a.s.a.p.
TDS P 200 Refrigerate Analyze a.s.a.p.

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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Container Min. sample Preservation Maximum
Determination size (mL) storage
TAN P 100 Refrigerate Analyze a.s.a.p.
-
NO2 P 100 Refrigerate Analyze a.s.a.p.
NO3- P 100 Refrigerate Analyze a.s.a.p.
3-
PO4 P 100 Refrigerate Analyze a.s.a.p.
SiO2 P 100 Refrigerate Analyze a.s.a.p.
Total iron G 125 Add 1mL HNO3 to 15 days
pH<2.
BOD DP 500 Refrigerate 24 hours
Chlorophyll-a DP 500 Refrigerate 24 hours
Note: P: plastic; G: glass; DG: dark glass, DP: dark plastic
3. Sampling procedure:

Select and clean the

containers

Collect the sample at 20 cm

in depth

Capped tightly, identify the

samples

Samples
preservation

Transfer the samples to the

laboratory

 Clean the bottle with tap water. Remove the dirt that may influence the quality of
sample.
 Sink the bottle into water body (see above figure)
 Fill the bottle. Carefully capped, taking care to prevent entry of dust that may
contaminate the sample.
 In case dissolved oxygen sampling, a small air space may influence the analytical
result.

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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4. Sample preservation
 In general, sample bottles should be resealed and stored in a clean, cool, dark
environment and protected from recontamination. Additional methods of preservation
include freezing, solvent extraction and the addition of chemical preservatives.
 Suggested chemical preservatives and recommended maximum storage times for
samples for various analyses are summarised in above table (item 2 – Sample
containers).
 The preservative treatment may be applied immediately on sampling or may already
be contained in the sample bottle.
5. Transfer and storage of samples
 The sample collection process should be co-ordinated with the laboratory. Analysts
need to know how many samples so that appropriate quantities of reagent chemicals
can be prepared.
 If sample bottles are provided by the laboratory, this ensures that they are of adequate
volume and have been properly prepared (with added chemical preservatives where
necessary).
Each sample bottle must be provided with an identification label on which the following
information is legibly:
 Name of the study.
 Site code
 Place of sampling (district, pond, river...)
 Date and time of sampling.
 Name of the individual who collected the sample.
Sample bottles should be placed in a box for transport to the laboratory.
If the delay between sample collection and bacteriological analysis will be less than 1 day,
samples should simply be kept in a cool, dark place.

Sample transport box

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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GRAVIMETRIC METHODS OF ANALYSIS
2540-D. TSS (Total Suspended Solids)
1. Method:
Gravimetric method
Method as Standard Method: 2540-D. TSS
2. Principle:
A well-mixed sample is filtered through a weighed standard glass-fiber filter and the
residue retained on the filter is dried to a constant weight at 103 to 105°C. The increase in
weight of the filter represents the total suspended solids.
3. Apparatus:
Glass-fiber filter 47, 0.45 µm; desiccator; drying oven, for operation at 103 oC to
105°C; analytical balance (capable of weighing to 0.00001 mg), graduated cylinder; filtration
apparatus; aluminum weighing dish; and others
4. Procedure:
4.1 Preparation of glass-fiber filter
- Heat glass-fiber filter at 103 to 105°C for 1 h.
- Store and cool dish in desiccator for 30 min.
- Weigh immediately.
- Mark the filter papers using a pencil
4.1 Sample analysis:
- Measure a volume of well-mixed sample
- Place a filter paper into the vacuum filtration apparatus, filter the water sample
- Carefully remove filter from filtration apparatus and transfer to an aluminum weighing
dish
- Dry for at least 1 h at 105 °C in an oven, cool in a desiccator for 30 min, and weigh.
- Repeat the cycle of drying, cooling, desiccating, and weighing until until the weight
change is less than 4% of the previous weight.
5. Calculation:
TSS (mg L-1 ) 
A - B  1.000  1.000
Where: VS mL 
VS (mL): volume of water sample filtered; A = weight of filter + dried residue (g), and B =
weight of filter (g)
6. Precision:
Single-laboratory duplicate analyses of 50 samples of water and wastewater were made
with a standard deviation of differences of 2.8 mg/L.

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VOLUMETRIC METHODS OF ANALYSIS
Free CO2
1. Methods:
Neutralize with NaOH
Standard Method: 4500-CO2 C.
2. Principle
Free CO2 reacts with sodium hydroxide to form sodium bicarbonate. Completion of the
reaction is indicated by the development of the pink color characteristic of phenolphthalein
indicator at pH of 8.3. NaOH + CO2  NaHCO3
3. Interferences
Cations and anions that quantitatively disturb the normal CO2-carbonate equilibrium
interfere with the determination. Metal ions that precipitate in alkaline solution, such as
aluminum, chronium, copper and iron contribute to high results. The titrimetric method for
CO2 is inapplicable to samples containing acid mine wastes and effluent from acid-generated
cation exchangers. Negative errors may be introduced by high total dissolved solids.
4. Preparation of chemicals
- Dung dịch NaOH 0.01N: Dilute 100 mL of 0.1N NaOH to 1.000 mL
- Phenolphthalein indicator solution: Dissolve 0.1 g of phenolphthalein (C20H14O4) in
100 mL of 95 percent ethyl alcohol.
5. Sampling and storage
Fill completely glass-stoppered bottle and do not let sample remain in contact with air
or be agitated. Keep the samples at low temperature for few hours. Make the laboratory
examination as soon as possible to minimize the effect of CO2 change.
6. Procedure
With a graduated cylinder, measure a 50 mL water sample into a 100-mL erlenmeyer
flask. Add 2 to 3 drops of phenolphthalein indicator solution. If sample turn pink, the pH is
above 8.3 and free carbon dioxide is absent. If the sample remains colorlesss, it contains free
carbon dioxide. Titrate with NaOH 0.01N solution to the first appearance of faint pink, which
remaining for 30 seconds.
7. Calculation
Free CO2 concentration is calculated as below:
Where:
V  N  44 1.000 - V: Volume of titrant
CO 2 (mg/L)  - N: Normality of titrant
VS
- VS: Volume of sample

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VOLUMETRIC METHODS OF ANALYSIS
DISSOVED OXYGEN (DO)
1. Method:
Winkler method (iodometric method).
Standard Method: 4500-O. B
2. Principle:
- It is based on addition of divalent manganese solition, follwed 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 high valency
states.
Mn2+ + 2OH- + ½ O2 = MnO2 + 2H2O
- In the presence of iodide ions in an acidic solution, oxidized manganese reverts to the
divalent state, with the liberation of iodine equivalent to the original DO content.
MnO2 + 2I- + 4H+ = Mn2+ + I2 + 2H2O
- The iodine is then titrated with a standard solution of thiosulfate. The titration end
point can be detected visually, with a starch indicator.
I2 + Starch-I2 (blue) + Na2S2O3  Na2S4O6 + NaI + H2O + Starch (colorless)
3. Interferences
Nitrite is the most common interference in biologically treated effluents.
4. Preparation chemicals:
- Manganous sulfate solution (MnSO4): Dissolve 50 gram MnSO4.5H2O or 41 gram
MnCl2.4H2O in distilled water, fill up to 100 mL.
- KI-NaOH solution: Dissolve 50 gram NaOH and 15 gram KI (or 14 gram NaI) in
distilled water, fill up to 100 mL.
- H2SO4 concentration (d=1,84, 98%) or H3PO4 concentration (d= 1,88).
- Standard Na2S2O3 0.01N titrant: Dilute 50 mL standard 0.1N Na2S2O3 with distilled
water, fill up to 500 mL.
- Starch 1%: Dissolve 1.0 gram (C6H12O6)n in 100 mL of boiled distilled water (~80-90
o
C). Mix well and add 10 drops of formaline. Store in amber bottle for up to 3 months.
5. Collection and preservation of samples
- Collect samples very carefully. Do not let sample remain in contact with air or be
agitated. No air bubble is entered.
- Samples are stored by adding:
 1 mL MnSO4
 1mL KI-NaOH (alkali-iodide solution)

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- Protect stored samples from strong sunlight and titrate as soon as possible.
6. Procedure
- After sampling and adding the chemicals, stand for 5-10 minutes
- Add 2 mL 98% H2SO4 concentration or H3PO4 concentration.
- Mix well.
- Pour 50 mL into a 50 mL-erlenmeyer.
 Titrate with 0.01N Na2S2O3 solution to a pale straw color. Add a few drops of
starch solution and continue titration to first disappearance of blue color.
 Write down the volume of standard Na2S2O3 0.01N titrant used.
7. Calculation

Vtb  N  8 1.000
DO (mg/L) 
Vm
Where:
- V: volume of standard Na2S2O3 0.01N titrant.
- N : concentration of Na2S2O3 (normally 0.01N ).
- 1,000: converted to liter.

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VOLUMETRIC METHODS OF ANALYSIS
TOTAL ALKALINITY
1. Method:
Acid titration method
Standard Method: 2320. Alkalinity
2. Principle:
Alkalinity is defined as a measure of the buffering capacity of water to neutralize strong
acid. This capacity is attributed to bases that are present in aquaculture pond waters including
HCO3-, and CO32-. High level of alkalinity in water means high buffering capacity.
In order to determine alkalinity, water is neutralized by sulfuric acid or hydrochloric
acid to a certain pH end point (pH 8.3 for phenolphthalein alkalinity and pH 4.4 for total
alkalinity), then the volume of the acid used for the titration is recorded. Phenolphthalein
indicator is used for alkalinity titration to pH end point 8.3. Methyl orange indicator is used
for pH end point 4.5. The reactions are as follows:
pH range  8.3
CO32- + H+ = HCO3-
pH range from 8.3 - 4.5
HCO3- + H+  H2O + CO2
3. Interferences
Suspended solids interfere by causing indistinct endpoint.
4. Preparation of chemicals:
- PRE 1: Pipet 27 mL of H2SO4 98% and dilute to 1.000mL with distilled water (DW).
- Phenolphtalein 1% indicator solution: dissolve 1.0g Phenolphtalein in 100mL C2H5OH
100%.
- Standard H2SO4 0.01N titrant: dilute 100 mL of 0.1N H2SO4 solution with distilled
water to 1,000mL.
- Methyl orange 0.1% indicator solution: dissolve 0.1 g methyl orange in 100mL
distilled water.
5. Collection and preservation of samples
Sample is collected in the plastic bottle and keep cold.
6. Procedure:
50mL in 100mL Erlenmeyer flask;
Add 3 drops Phenolphtalein 1% indicator solution, if:
+ The solution becomes pink color: (pH >= 8.34): add H2SO4 0.01N solution titrant
slowly (by Burette), with continuous stirring, until the last pink colour disappears.

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Add the last 3 drops Methyl orange, continuously add H2SO4 0.01N solution titrant.
At the end point the solution normally is orange, write down volume (P) of 0.01N
H2SO4 titrant.
+ Solution is colourless (pH value of sample <8.34): Add the last 3 drops methyl
orange, add H2SO4 0.01N solution titrant slowly. At the end point the solution
normally is orange, write down volume (T) of H2SO4 0.01N titrant.
7. Calculation:
Vp  N  501.000
P (mgCaCO 3/L) 
Vs
Vt  N  50 1.000
T (mgCaCO 3 /L) 
Vs
Where:
V: volume of H2SO4 0.01N solution (mL) used
N: concentration of H2SO4 (0.01N)

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VOLUMETRIC METHODS OF ANALYSIS
2340. HARDNESS
1. Method :
Complexon titration
Standard Method : 2340C. EDTA Titrimetric Method
2. Principle:
The concentration of calcium plus magnesium expressed as equivalent calcium
carbonate traditionally has been as a measure of total hardness. A small amount of a dye such
as Eriochrome Black T is added to an aqueous solution containing calcium and magnesium
ions at a pH of 10, the solution become wine red. If EDTA is added as a titrant, the calcium
and magnesium will be stably complexed (CaEDTA & MgEDTA), and when all of the
magnesium and calcium has been complexed the solution turns from wine red to blue,
marking the end point of titration.
M2+ + M-E.B.T + Na2H2Y  Na2MY + 2H+ + E.B.Tfree
(Wine red) (Blue)
3. Interferences
Some metal ions interfere by causing fading or indistinct end point or by stoichiometric
consumption od EDTA. Suspended or colloidal organic matter also may interfere with the
end point.
4. Reagents
- pH10 buffer: Dissolve 67 g ammonium chloride (NH4Cl) in 570 mL concentrated
ammonium hydroxide (NH4OH).
- Standard solution of 0.01N EDTA: Dilute 100 mL of 0.1N EDTA stock solution
with distilled or deionized water to 1000 mL.
- Eriochrome Black T indicator: Dry 0,5 g Eriochrome Black T and 100 g NaCl at
110 oC and cool in a desiccator, then pulverize them into fine powder.
4. Sampling and storage
Sample is collected in a polyethylene or a borosilicate glass bottle and stored at a low
temperature and analyze sample in few days.
5. Procedure
- Dilute the sample if necessary. Select dilution rate that requires less than 10 mL EDTA
titrant.
- With a graduated cylinder, measure a 50 mL water sample into a 100-mL erlenmeyer
flask. Add 1 mL of buffer solution and mix. Add an appropriate amount of dry-powder
indicator formation, the sample will become wine red if it contains calcium or
magnesium ions. Titrate with EDTA standard solution, at the end point solution will

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change from wine red to pure blue. Calculate the total hardness (H) from equation
below:
7. Calculation
Calculate the total hardness with equation:
Vt  N  50 1.000
H (mg CaCO 3 /L) 
Where: Vs
H = Total hardness, expressed as mg CaCO3/L
V = Volume of EDTA standard solution required to titrate the sample to the end point (mL).
N = Normality of EDTA standard solution titrant.
VS = Sample volume in mL

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SPECTROPHOTOMETRIC METHODS OF ANALYSIS
TOTAL AMMONIA NITROGEN (TAN; NH4+/NH3)
1. Method:
Standard Method: 4500-NH3 F. Phenate Method
2. Principle:
An intensely blue compound, indophenol, is formed by the reaction of ammonia,
hypochlorite, and phenol catalyzed by sodium nitroprusside. Both ammonia and ammonium
are measured, because all of ammonium are converted to ammonia in a strong alkaline
condition. Thus, the result are expressed as total ammonia nitrogen (TAN).
NH3 + ClO- NH2Cl + OH-

NH2Cl + 2 OH + 2ClO- O N OH + 3HCl + 2OH-

+
O N OH O N O- + H
Indophenol

3. Interferences:
Complexing magnesium and calcium with citrate eliminates interference produce by
precipitation of these ions at high pH. Remove suspended solid interference by pressure
filtration.
4. Preparation of chemicals:
- Phenol solution (Reagent A): Mix 11.1 mL of C6H5OH (≥89%) with ethyl ethanol to
a final volume of 100 mL. Prepare weekly.
(Caution: Wear gloves and eye protection when handling phenol; use good ventilation
to minimize all personnel exposure to this toxic volatile subtance).
- Sodium nitroprusside, 0.5% w/v (Reagent B): Dissolve 0.5 g sodium nitroprusside
- Na2[Fe(CN)5NO].2H2O (sodium nitroferricyanide) with 100 mL deionized water.
Store in amber bottle for up to 1 month.
- Alkaline citrate: Dissolve 200 g trisodium citrate and 10 g NaOH in deionized water.
Dilute to 1000 mL.
- Sodium hypochlorite: a commercial product ~5%.
- Oxidizing solution (Reagent C): Mix 100 mL alkaline citrate solution with 25 mL
sodium hypochlorite. Freshly Prepared
- Stock ammonium solution (1000 mg/L): Dissolve 3.819 g anhydrous ammonium
chloride or 4.717 g anhydrous ammonium sulfate in deionized water, and dilute to
1000 mL.
- Standard ammonium solution (10 mg/L): Dilute 10 mL with deionized water to 1000
mL.

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5. Sampling and storage:
Sample should be analyzed within 24 h of collection, refrigerate unacidified at 4 oC. For
reservation up to 28 days, freezed at -20 oC
6. Procedure
- Prepare a blank and a series of standard solutions, including the concentrations of 1,
0.5, 0.25, 0.125 and 0.0625 ppm.
1 mL 5 mL 5 mL 5 mL 5 mL

9 mL 5 mL 5 mL 5 mL 5 mL Discharge 5 mL
DW DW DW DW DW 5 mL DW

Std.
10 ppm

1 ppm 0.5 ppm 0.25 ppm 0.125 ppm 0.0625 ppm 0 mg/L

- To a 5-mL water sample or series of standard solutions in a 25-mL test-tubes.

- Add, with thorough mixing after each addition, 4 drops of phenol solution (A), 2
drops of sodium nitroprusside solution (B), and 8 drops of oxidizing solution (C).
- Cover samples with plastic wrap or parafin wrapper film. Let color develop at room
temperatue in subdued light for at least 1 h.
- Measure absorbance at 630 nm (freshwater) or 640 nm (brackishwater).
7. Calculation
Prepare a standard curve by plotting absorbance readings of standards against ammonia
concentrations of standards. Compute sample concentration by comparing sample absorbance
with the standard curve, as follow:
Ab
A = aC + b  C
a
Where:
A = Absorbance ; C = Concentration (ppm or mg/L) ; a, b = coefficient of standard curve
Caution: The concentration of sample from above calculation is total ammonia nitrogen
(TAN), including ionized ammonia nitrogen (NH4+-N) and unionized ammonia ntrogen (NH3-
N). Base on pH and temperature value at sampling time, calculate unionized ammonia
nitrogen concentration following the equation below:

TAN
NH3 − N = 2729.92
(0.09018+ o )−pH
1+10 273.15+t

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Rate of % NH3 in pH and Temperature dependence

Temperature (oC)
pH 16 18 20 22 24 26 28 30 32
7.0 0.30 0.34 0.40 0.46 0.52 1.60 0.70 0.80 0.92
7.2 0.47 0.54 0.63 0.72 0.82 0.95 1.10 1.27 1.00
7.4 0.74 0.56 0.99 1.14 10.30 1.50 1.73 2.60 2.36
7.6 1.17 1.35 1.56 1.79 2.05 2.35 2.72 3.13 3.96
7.8 1.84 2.12 2.45 2.80 3.21 3.65 3.75 4.88 5.72
8.0 2.88 3.32 3.83 4.37 4.99 5.71 6.55 7.52 8.75
8.2 4.49 5.16 5.94 5.76 7.68 8.75 10.00 11.41 13.22
8.4 6.93 7.94 9.09 10.30 11.65 13.20 14.98 16.96 19.46
8.6 10.56 12.03 13.68 15.40 17.28 19.42 21.83 24.45 27.66
8.8 15.76 17.82 20.08 22.38 24.88 27.64 30.68 33.90 37.76
9.0 22.87 25.57 25.47 31.37 34.42 37.71 41.23 44.84 49.02
9.2 31.97 35.28 38.65 42.01 45.41 48.96 52.65 56.30 60.38
9.4 42.68 46.32 50.00 53.45 56.86 60.33 63.79 67.12 70.72
9.6 54.14 54.77 61.31 64.54 67.63 70.67 73.63 76.39 79.29
9.8 65.17 68.43 74.53 74.25 76.81 79.25 61.57 83.68 85.85
10.0 74.78 77.46 79.92 82.05 84.90 85.25 87.52 89.05 90.58
10.2 82.45 84.48 86.32 87.87 89.27 90.56 91.75 92.80 93.84

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SPECTROPHOTOMETRIC METHODS OF ANALYSIS
NITRITE (N-NO2-)
1. Method:
Diazonium method
Standard Method: 4500-NO2- B. Colorimetric Method
2. Principle:
Nitrite (NO2-) is determined through formation of a reddish purple azo dye produced at
pH 2.0 to 2.5 by coupling diazotized sulfanilamide with N-(1-naphthyl)-ethylenediamine
dihydrochloride (NED dihydrochloride)
- Reaction:

NO2- + HO3S NH2 + 2H+

Acid sulfanilique
+
HO3S N N + 2H2O

Diazonium sulfanilique

OH OH

+
HO3S N N +

HO3S SO3H
. napthylammine

OH OH

N N SO3H + H+

HO3S SO3H

3. Interferences:
Nitrogen trichloride (NCl3) impacts a false red color when color reagent is added. The
following ions interfere because of precipitation under test conditions. Cupric ion may couse
low results by catalyzing decomposition of diazonium salt. Suspended solids cause possitive
interference, remove suspended solid interference by vacuum filtration.

4. Apparatus
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- 1-5 mL Micropipettes
- Spectrophotometer, cuvette 1 cm or greater
5. Preparation of chemicals
- Color reagent: To 800 mL deionized water add 100 mL 85% phosphoric acid and 10
g sulfanilamide. After dissolving sulfanilamide completely, add 1 g N-(1-naphthyl)-
ethylenediamine dihydrochloride. Mix to dissolve, then dilute to 1000 mL with
deionized water. Solution is stable for about a month when stored in a dark bottle in
refrigerator.
- 10 mg/L N-NO2-: mix 10 mL of 100 mg/L N-NO2- with deionized water to 100 mL.
6. Collection and preservation of samples
Most reliable results are obtained on fresh samples. Samples should analyzed within 1
to 2 d of collection, refrigerate unacidified a 4 oC or freeze at -20 oC.
7. Procedure:
- Prepare a blank and a series of standard solutions comprising the concentrations of 0.8,
0.4, 0.2, 0.1 and 0.05 mg/L
1 mL 5 mL 5 mL 5 mL 5 mL

9 mL 5 mL 5 mL 5 mL 5 mL Remove 5 mL
DW DW DW DW DW 5 mL DW

Std.
8 mg/L

0.8 mg/L 0.4 mg/L 0.2 mg/L 0.1 mg/L 0.05 mg/L 0 mg/L

- To a 5-mL water sample or series of standard solutions in a 25-mL test-tubes.

- Add, with thorough mixing after addition, 5 drops of color reagent.
- Wait for 5-10 min
- Read at 543 nm using the spectrophotometer
8. Calculation
Prepare a standard curve by plotting absorbance readings of standards against nitrite
concentrations of standards. Compute sample concentration by comparing sample absorbance
with the standard curve (see equation below).
A = aC + b  C  Ab
a

Where: A = Absorbance; C = Concentration (ppm or mg/L); a,b = coefficient of standard

curve.

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SPECTROPHOTOMETRIC METHODS OF ANALYSIS
ORTHO-PHOSPHATE (PO43-)
1. Method:
Standard Method: 4500-P D. Stannous Chloride (SnCl2) Method
2. Principle:
Ammonium phosphomolybdate is formed by the reaction of orthophosphate in a strong
acid solution, then reduced by stannous chloride to intensely colored molybden blue.
7H3PO4 + 12(NH4)6Mo7O24.4H2O → 7(NH4)3[PO4(MoO3)12] + 51NH4+ + 51OH‐ + 33H2O
(Ammonium phosphomolybdate)
(NH4)3[PO4(MoO3)12] + Sn2+ + 16H+ = (NH4)3[PO4(4MoO2.2MoO3)2] + Sn4+ + 8H2O
3. Interferences
SiO2 and AsO43- cause positive interferences when sample water is heated. AsO43-, F-,
thorium (Th), bismuth (Bi), sulfide, thiosulfate and thiocyanate cause negative interferences.
Suspended solids cause possitive interference, remove suspended solid interference by
vacuum filtration.
4. Preparation of chemicals:
- Strong acid solution: Slowly add 300 mL conc H2SO4 to about 600 mL distilled water.
When cool, add 4.0 mL conc HNO3 and dilute to 1 L.
- Solution A: Ammonium molybdate solution: Dissolve 25 g (NH4)6Mo7O24⋅ 4H2O in
175 mL distilled water. Cautiously add 280 mL conc H2SO4 to 400 mL distilled water.
Cool, add molybdate solution, and dilute to 1000 mL.
- Solution B: Stannous chloride solution: Dissolve 2.5 g fresh SnCl2⋅2H2O in 100 mL
glycerol. Heat in a water bath and stir with a glass rod to hasten dissolution. This
reagent is stable and requires neither preservatives nor special storage.
- Standard phosphate solution (PO43--P, 10 mg/L): Dilute 20 mL of 500 mg/L PO43- -P
with distilled water to 1000 mL.
5. Apparatus:
- 1-5 mL Micropipettes; spectrophotometer; 1 cm-cuvette or greater
6. Sampling and preservation:
Most reliable results are obtained on fresh samples. If samples are analyzed within 24 h
of collection, refrigerate at 4 oC. For reservation up to 28 days, freeze at -20 oC.
7. Procedure:
- Prepare a blank and a series of standard solutions, including 1, 0.5, 0.25, 0.125 and
0.0625 ppm.

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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1 mL 5 mL 5 mL 5 mL 5 mL

9 mL 5 mL 5 mL 5 mL 5 mL Remove 5 mL
DW DW DW DW DW 5 mL DW

Std.
10 mg/L

1.0 mg/L 0.5 mg/L 0.25 mg/L 0.125 mg/L 0.0625 mg/L 0 mg/L

- To a 5-mL water sample or series of standard solutions in a 25-mL test-tubes.

- Add, with thorough mixing after addition, 6 drops of ammonium molybdate solution
and 1 drop of stannous chloride solution.
- Measure absorbance of molybden blue solution at 690 nm between 5 to 10 minute after
adding reagents.
8. Calculation:
Prepare a standard curve by plotting absorbance readings of standards against phosphate
concentration of standards. Compute sample concentration by comparing sample absorbance
with the standard curve (see equation below).
Ab
A = aC + b  C
a
Where: A = Absorbance; C = Concentration (ppm or mg/L); a,b = coefficient of standard
curve.

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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SPECTROPHOTOMETRIC METHODS OF ANALYSIS
HYDROGEN SULFIDE (H2S)
1. Method:
Standard Method: 4500-S2- D. Methylene Blue Method
2. Principle:
Methylene blue method is based on the reaction of sulfide, ferric chloride (FeCl 3) and
dimethyl-p-phenylenediamine to produce methylene blue. Ammonium phosphate is added
after color development to remove ferric chloride color. Both H2S gas and HS- ion are
measured. All HS- ion are converted to H2S in strong acid condition, the procedure thus gives
an estimate of total sulfide.

3. Interference
Sulfite (SO32-) slows down the reaction producing methylene blue if its concentration is
greater than 10 mg/L. Raise ferric concentration to 2-6 times to prevent sulfite interference.
Suspended solid cause positive interference, remove suspended solid interference by pressure
filtration.
4. Preparation of chemicals
- Solution A: Amine-acid reagent: dissolve 6.25 g C8H14Cl2N2 với 1+1 HCl or H2SO4
to 1000 mL.
- Solution B: Ferric chloride solution: dissolve 100 g FeCl3.6H2O with 40 mL distil
water.
- Solution C: Diammonium hydrogen phosphate solution: Dissolve 40 g (NH4)2HPO4
with 80 mL distil water.
- Sulfide standard solution (10 mg/L): mix 1 mL of 1000 mg/L S2- with free-oxygen
distil water to 100 mL.
5. Apparatus:
- 1-5 mL Micropipettes
- Spectrophotometer
- 1 cm-cuvette or greater
6. Sampling and storage:
Collect water samples with minimum aeration. Either analyze samples immediately after
collection or preserve with zinc acetate solution for later analysis. To preserve a sample for a
total sulfide determination put zinc acetate and sodium hydroxide solutions into sample bottle

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
<>< <>< <><
before filling it with sample. Use 0.2 mL 2M zinc acetate solution per 100 mL sample. The
final pH should be at least 9. Add more NaOH if necessary. Fill bottle completely and stopper.
7. Procedure:
- Prepare a blank and a series of standard solutions, including 1.0, 0.5, 0.25, 0.125 and
0.0625 ppm.

1 mL 5 mL 5 mL 5 mL 5 mL

9 mL 5 mL 5 mL 5 mL 5 mL Remove 5 mL
DW DW DW DW DW 5 mL DW

Std.
10 mg/L

1.0 mg/L 0.5 mg/L 0.25 mg/L 0.125 mg/L 0.0625 mg/L 0 mg/L

- To a 5-mL water sample or series of standard solutions in a 25-mL test-tubes.

- Add, with thorough mixing after each addition, 2 drops of Solution A, then 1 drop of
Solution B.
- Waite 3 to 5 min, add 1 drop of Solution C to remove the color of remaining ferric
chloride solution.
- Measure absorbance at 664 nm (roundly 15 min).
8. Calculation:
Prepare a standard curve by plotting absorbance readings of standards against sulfide
concentrations of standards. Compute sample concentration by comparing sample absorbance
with the standard curve (see equation below).
Ab
A = aC + b  C
a

Where: A = Absorbance ; C = Concentration (mg/L or ppm); a, b = coefficient of standard

curve
Caution: The concentration of sample from above calculation is total sulfide, including
H2S gas and HS-ion. Base on pH and temperature value at sampling time, calculate H2S gas
concentration following the equation below:
HS-
H2 S= 5765.4
10[pH-(273.15+T)-15.0455× ln(273.15+T)+98.08]

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 Modern Analytical Chemistry- A Practical Guide to the Sample Collection, Preservation and Analysis
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LABORATORY REPORT

Name of student (st code)

1. .................................................................
2. ................................................................. Group:
3. .................................................................
Subgroup:
4. .................................................................
5. ................................................................. Time:
6. .................................................................

Parameter Unit Method for Results Optimal range

analysis in aquaculture
TSS mg L-1
CO2 mg L-1
DO mg L-1
Alkalinity mg CaCO3 L-1
Hardness mg CaCO3 L-1
N-NO2- mg L-1
TAN mg L-1
P-PO43- mg L-1
Total S2- mg L-1

Note: Subgroups are required to submit this report before lab practical final exam.

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