SCHOOL OF MARINE & ENVIRONMENTAL

SCIENCES
KIM4702 Introduction to Environmental Analysis
Practical
Semester 2, 2016/17
Laboratory Manual Version 3 (15 March 2016)
 Table of Content

Safety Rules..........................................................................................................2

Introduction to KIM4702 Practical Work.............................................................4

Practical 1- Introduction to Water and Air Sampling............................................5

Practical 2- Analysis of Dissolved Oxygen (DO).................................................7

Practical 3- Determination of Biochemical Oxygen Demand (BOD)................18

Practical 4- Determination of Chemical Oxygen Demand (COD).....................23

Practical 5- Determination of Ammonia in water with Phenate Method............28

Practical 6- Determination of Selected Metals in Suspended Particulate...........34

Practical 7- Determination of Selected Ionic Species in Particulate Matter 10m

(PM10) and rain water....................................................................40

5
 Safety Rules
Safety glasses and laboratory coats must be worn at all times in
laboratories where chemicals are stored and in use.

Closed shoes must be worn at all times in laboratories. Do not wear

thongs and open sandals.

Long hair must be tied back.

Eating and drinking in the laboratory are strictly prohibited.

Unauthorized visitors are NOT permitted to enter the laboratory.

Learn the location and proper use of safety showers and eyewashes.

Avoid skin contact with chemicals; wash your hands after every practical
session.

Students are NOT permitted to take chemicals out of the laboratory.

Keep your work area neat and orderly. Clean up chemical and water spills
at once, seek from your demonstrator or lab staff as necessary.

The use of hand phone, personal audio or video equipment is prohibited

in the laboratory unless for teaching and learning purposes.

Risk Assessment must be carried out for each experiment before

coming to the practical session, and the complete forms signed by a
demonstrator, before work commences. The complete risk assessment
form must be submitted with the signed Practical Report Cover Sheet for
each experiment report.

6
THINGS TO BRING
Every student should have

a marker pen for labelling

a small towel or tissue papers
stationeries such as note-book for writing down data & observations or
reference about the experiment; a ruler and a calculator

APPARATUS

All of the apparatus prepared are in good condition. Every student must keep
the table tidy and clean including all the apparatus. The instructor or the
laboratory staff must be told about any damage done. After completing your
experiment, make sure that everything is clean, including your table and the
sink. Your table should be wiped dry.

CHEMICALS

The chemicals to be used are usually placed in the laboratory use it carefully
and avoid contamination. Some of the hazardous chemicals are kept in the
fume hood. Different droppers must be use for different chemicals. After
using the dropper, place, it back in the bottle that contained the chemicals.
Do not mix the droppers to avoid contamination of chemicals.

DO NOT TAKE OR USE ANY CHEMICAL MORE THAN IS NEEDED!

INSPECTION

The instructor will give briefings about the experiment at the beginning of
the laboratory class. Even though this is done, the student himself must read,
understand and be ready with a flow chart of the method or a summarized
method. If any problem arises about the theory or the method, consult your
instructor. Generally, the laboratory supervision, preparation of the
experiments is the responsibilities of the laboratory staff.

7
 Introduction to KIM4702 Practical Work
As clear stated in the Universiti Malaysia Terengganu Undergraduate Handbook
(http://www.umt.edu.my/v10/doc/Peraturan_Akademik_UMT.pdf), full practical
attendance throughout semester is compulsory in order to sit for final
examination.

Course Synopsis
Topics in this course will cover discussions on transport of pollutants in the
environment, ecological and health effects of chemical pollution, environmental
monitoring strategies and analysis, and quality control and assurance in
environmental analysis. The practical in this course involved environmental
sampling, environmental sample preparation and analysis in laboratory.

Course Learning Objectives

1. To learn different analytical methods in the environmental sample
determination.
2. To learn and understand the basic design of environmental sampling.
3. To learn and practice data manipulation and interpretation for reporting.
4. To learn and practice write a scientific laboratory report.

Course Grading System

Assessment Percentage Mark Grade

Practical Report 20% 80-100% A
Lab Test 10% 75-79% A-
Assignments 10% 70-74% B+
Test 20% 65-69% B
Final Exam 40% 60-64% B-
Total 100% 55-69% C+
50-54% C
45-49% C-
40-44% D
39% or less F

8
 Practical 1- Introduction to Water and Air
Sampling
Purpose

To conduct water sampling and PM10 sampling in UMT campus

Objectives

To perform a risk assessment of a field-based activity

To introduce application of laboratory Chain of Custody form

To learn good practices for collecting water and PM10 samples and performing
in-situ measurement

Revision/Reading

KIM3701 and KIM4701

Introduction

PART I (Air Particulate or PM10 Mass Sampling) - The PM10 standards are
expressed as a weight of PM10 particles per volume of air (micrograms per
cubic meter). PM10 mass is collected using a high volume sampler (40 cubic
feet per minute) and a quartz fiber filters (8" x 10"). The standards do not
consider the size distribution or the chemical make-up of the particles, although
these are important factors in terms of control strategies and of the health risks
associated with PM10. High and low volume air samplers are instruments used
to collect samples of air particles. The difference between high and low volume
air samplers is the amount of air sampled. High volume air samplers typically
sample more than 1500 cubic metres (m3) of air over a 24-hour period, while
low volume air samplers draw through only 24 m3 of air, or less.

Particles smaller than 10m are especially concerning as these particles can
enter the human respiratory system and penetrate deeply into the lungs, causing

9
adverse health effects. Motor vehicles and other combustion processes that burn
fossil fuels such as power stations, industrial processes and domestic heaters,
generate PM10. Dust storms and smoke particles from bushfires can also be
another source of PM10 missions.

Instruments used to measure PM10 are either a high or low volume air sampler.
The PM10 high volume air sampler is similar to Figure 1. The size selective
inlet removes particles larger than 10m by using their greater inertia to trap
them on a greased plate, while smaller particles pass through the instrument
onto the pre-weighed filter.

Figure 1: Diagram of PM10 sampler

Part II (Characterization of Surface water in UMT Campus) Surface water

resources such as natural rivers and lakes support a wide variety of human
livehood and ecological species. Protecting water from contamination became

10
paramount in view of the country is undergoing rapid development since 1980s.
In 1974, the Environmental Quality Act (1974) was implementing to control and
prevent environmental pollutions. The enactment of law is to preserve and
improve the water quality status of water bodies, pollution sources that input
various constituents into the watercourses need to be appropriately managed and
mitigated (control at source). Pollution sources are divided into two main
categories; point and non-point sources of pollution and is characterized by their
physical, chemical and biological attributes where some common constituents
are listed in Table 1.

Table 1: Water Quality Constituents

Physical Chemical Biological

Total Suspended Solid Dissolved Oxygen Total Coliform
Total Dissolved Solid Biological Oxygen Fecal foliform
Demand
Turbidity Chemical Oxygen Escherichia coli
Demand
pH Total Organic Carbon Phytoplankton
Ammoniacal Nitrogen Chlorophyll (algae)
Nitrite & Nitrate Macroinvertabrates
Phosphorus
Heavy metals
Organic Pollutants

In this practical, you and your team members will collect PM10, COD and
Ammoniacal Nitrogen sample for Practical 4-6.

Checklist for water sampling

1. Identify surface water bodies found within UMT campus. Highlight the
type of surface water bodies in different colors in Figure 2. Label your
figure.

11
Figure 2 Surface water bodies within UMT Campus

12
2. Prepare a Sampling + laboratory chain of custody for your water sample.
You need this form during the sampling day and keep the form until end
of the analysis (Tips: your C&C form should include critical information
such as as sample holding time, sample preservation ,volume, sampling
time, analysis time, etc.)
3. Prepare sample bottles, preservatives, label and marker pens, sample
storage/transit containers and ice packs. Dont forget to bring field note,
risk assessment form and C&C forms for documentation.
4. Check and calibrate meter; Lab Demonstration (pH, conductivity,
dissolved oxygen, turbidity, thermometers).

For sampling only:

1. At each site, record the sampling time, pH, DO and salinity data.

2. Collect COD and Ammoniacal Nitrogen sample.

3. Filtere the Ammoniacal Nitrogen sample ONLY using 0.45m membrane

cartridge.

4. The UNFILTERED COD sample will be preserved with 1ml of concentrated

H2SO4.

5. Complete your C&C form and bring the samples back to Laboratory.

6. Keep the COD samples in designation location until further analysis (ask lab
assistant for the location, unlabeled sample will be discarded).

7. Keep the Ammoniacal Nitrogen sample in freezer until further analysis (ask
lab assistant for the location, unlabeled sample will be discarded).

Checklist for PM10 sampling

1. Calibrate high volume air samplers and enter calibration data into Sample
volume sheet (Lab Demonstration).

13
2. Determine predominant wind direction (e.g., upwind, downwind, or near
point source) by reviewing data from the nearest meteorological stations.

3. Sketch the permanent monitoring location.

4. Record the weight of filter paper (value will be given)

For sampling only:

1. Demonstration of PM10 sampling

Discussion guide

Water Sampling

1. In your report, you need to report pH, Salinity, Temperature and

dissolved oxygen values in Pond A, B, C and D using a table.
2. Plot pH, salinity, DO and temperature in Pond A-D using line or bar
graph/graphs (excel). Based on your graphs discuss the trend of
parameters in all four ponds. Explain why some of the parameter values
vary as a function of sampling time.
3. Attached your sampling holding information, C&C form and colored
sampling map in the report.

Air Sampling

If you are requested by supervisor to conduct an environmental monitoring

within UMT campus, where will you put your PM10 air sampler? Explain why
you select that particular area. (You can use UMT map to help you to illustrate
the location of your sampling point).

14
 Practical 2- Analysis of Dissolved Oxygen (DO)

Purpose
To determine the DO of samples
To determine the DO at different temperatures and salinities

Objectives
To investigate the relationship between DO and temperature
To investigate the relationship between DO and salinity
To introduce the titration method for the determination of DO

Revision/Reading
KIM3701 and KIM4701

Introduction

All aquatic animals is dependent on the presence of dissolved oxygen for their
daily activities. A healthy river requires the presence of oxygen for the whole
ecosystem, and hence this parameter is used as one of the water quality
indicators. Modified Winkler method is applied in this practical to determine the
dissolved oxygen in the samples. The end point is detected using starch by
observing the disappering of blue colour. Divalent manganese solution and
strong alkali that are added to the sample precipitates the divalent manganese to
manganese(IV) oxide. When the sample is added with iodide solution and
acidified, the managenese(IV) ion is reduced to managanese(II). The iodine
produced in this step will be reacted with the thiosulfate standard solution.

The reduction of oxygen content in natural water can always be increased

through photosinthesis and aeration. Production of oxygen is high in day time
15
due to the photosynthesis. Dissolution of oxygen is dependent on the pressure,
temperature and salinity of the water. When the temperature increases, dissolved
oxygen is decreased as the dissolution of oxygen is exothermic process. In
addition, the dissolution of oxygen decreases with the increasing of water
salinity (electrolite).

Apparatus and Equipment

Beaker, 600 mL Erlenmeyer flask, 250 mL

Air pump Conical flask, 1 L
Burette, 50 mL Pipette, 1, 50 mL
Thermometer COD sampling bottles
Magnetic stir bar Stirring hotplate
DO meter

Chemicals (Prepared)

1 Manganese sulphate (Winklers 1): Dissolve 480 g of MnSO4.H2O and

dilute to 1 L of distilled water.

2 Iodide test solution (Winklers 2): Dissolve 500 g of NaOH and dilute
to 500 mL of distilled water. Dissolve 300 g KI in 450 mL of distilled
water. Mix both solutions well. Heat the resulting solution if necessary.
3 0.1% startch indicator :Dissolve 2 g of starch in 800 mL of distilled
water. Add 40% NaOH drop by drop into the solution until the solution
becomes clear. Add HCl until the solution is acidified by checking
with litmus paper. Then add 1 mL of glacial acetic acid into the
solution to preserve it. Dilute the solution to 1 L with distilled water.
4 0.01 N thiosulfate standard solution: Dissolve 2.483 g of sodium
thiosulfate (Na2S2O3.5H2O) and dilute to 1 L of distilled water.

16
5 Concentrated sulfuric acid (Winklers 3)

6 0.1 M sodium chloride: Dissolve 5.85 g of NaCl and dilute to 1 L of

deionized water.

Attention: Part A and Part B is two separate experiment. You can

coordinate among your group members to run the experiment
simultaneously
Procedure

A1. Sampling

1 Flush the sampling bottles twice with the samples.

2 Direct the sample into the bottom of bottle with a rubber tube. Ensure
the end of the tube is positioned exactly on the bottom surface to avoid
mixing of the sample.
3 Allow the sample overflow from the bottle for few times to avoid the
formation of the bubbles in the sample.
A2. Determination of F Factor

1 Fill the 1 L conical flask with 1 L of distilled water. Aerate the water
(~15 min) until it is saturated with oxygen.
2 Measure the temperature of the water and pour the sample slowly into
a sample bottle. Perform steps A3.1-A3.4 from the procedure of
determination of dissolved oxygen.
3 Pour 50 mL of the sample from the bottle into an Erlenmeyer flask.
4 Titrate the sample with thiosulfate standard solution as step A3.5.
5 Calculate the F factor.
mg oxygen/ L
Factor F=
0.1006 v 16

Where, V = mL thiosulfate standard solution

mg oxygen/L = Refer to the Table 1, where the oxygen content is

17
 temperature dependence
A3. Determination of Dissolved Oxygen in Samples.

1 Fill the sample bottle with the sample as described in the A1 sampling
procedure.
2 Add 1 mL each of the Winklers 1 and 2 reagents into the sample.
Stopper the bottle immediately and shake the bottle until the
precipitation is formed evenly. Ensure no bubbles are trapped in the
bottle.
3 Allow the precipitation forms at least 1/3 from the bottom and the
supernatant stays clear at the top layer.
4 Add 1 mL concentrated sulphuric acid to the bottle. Stopper and shake
the bottle until the precipitation dissolves. Ensure there is no bubbles
are trapped in the bottle.
5 Transfer 50 mL of the sample into an Erlenmeyer flask and titrate the
sample with thiosulfate standard solution until faint straw colour is
obtained. Add 1 mL of starch indicator and continue the titration until
the sample becomes colourless.
6 Determine the oxygen content in the sample using the following
formulae:

Oxygen content (mg/L) = 0.1006 x F x V x 16

B1. Determination of DO at Different Temperatures

1 Pour 300 mL of deionized (DI) water into a 600 mL beaker.

2 Place a magnetic stir bar into the beaker and stir the DI water on a
stirring hotplate. Ensure the stir bar is positioned at the centre of the
beaker to mix well the DI water.
3 Aerate the DI water and measure the DO with a DO meter. Ensure the
temperature and DO probes are positioned under the water.
4 Write down the DO and temperature when the DO reading becomes
stable. Remove the 600mL beaker from hot plate.
5 Carefully transfer water and magnetic stir bar into a tall drinking glass.

18
 Make sure the temperature and DO probe are under the water.
6 Heat the DI water to ~35 C and turn on the stirring. Write down the
temperature and DO when the temperature is reached.
7 Repeat step B1.6 for temperature ~45 C.
8 Remove the glass from heat, cool the water sample with ice cubes
water bath (using 600mL beaker) and repeat step B1.5 for temperature
~15 and 5 C.
B2. Determination of DO at Different Salinities

1 Pour 250 mL of 0.1 M NaCl solution into a 600 mL beaker.

2 Repeat steps B1.2-B1.4 for this solution.
3 Dilute the 0.1 M NaCl stock solution into 0.01, 0.001 and 0.0001 M
with 250 mL volumetric flask. Measure the DO and temperature by
repeating the steps B2.1-B2.2.
B3. Determination of DO in Samples

1 Pour 250 mL of lake water sample into a 600 mL beaker. Stir the
sample and measure its DO and temperature using DO meter.
2 Repeat the step for sea water sample.

Data and Calculations

Table 1 : Maximum DO concentrations vary with temperature.

Temperature (oC) DO (mg/L) Temperature (oC) DO (mg/L)

0 14.60 23 8.56

1 14.19 24 8.40

2 13.81 25 8.24

3 13.44 26 8.09

4 13.09 27 7.95

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 5 12.75 28 7.81

6 12.43 29 7.67

7 12.12 30 7.54

8 11.83 31 7.41

9 11.55 32 7.28

10 11.27 33 7.16

11 11.01 34 7.16

12 10.76 35 6.93

13 10.52 36 6.82

14 10.29 37 6.71

15 10.07 38 6.61

16 9.85 39 6.51

17 9.65 40 6.41

18 9.45 41 6.41

19 9.26 42 6.22

20 9.07 43 6.13

21 8.90 44 6.04

22 8.72 45 5.95

1. Collect and report all necessary data from this experiment in appropriate
forms under Results.

2. Calculate the F factor.

3. Calculate the oxygen content in the samples with the modified Winkler
method.

20
 4. Compare the oxygen contents obtained from both titration and DO meter.

5. Plot graphs of DO (ppm) versus temperature (C).

Discussion guide

1. What is the rational of performing A1.2 step during sampling?

2. What is the rational of performing A3.5 step, where starch is not added
one shot in the beginning of titration?

3. Discuss the effect of different temperatures and salinities of samples in

the determination of DO.

4. Discuss the results of DO obtained from samples. Relate with the

pollution level.
5. Discuss the precaution steps in sampling and determination of DO.
6. Support your discussion with appropriate references.

Practical 3- Determination of Biochemical

Oxygen Demand (BOD)
Purpose

To determine the BOD5 of samples

Objectives

21
To introduce the method for the determination of BOD5

Revision/Reading

KIM3701 and KIM4701

Introduction

Biochemical oxygen demand (BOD) is the amount of dissolved oxygen needed

by aerobic biological organisms to break down organic material present in water
sample at certain temperature over a specific time period. The BOD value is
most commonly expressed in milligrams of oxygen consumed per litre of
sample during 5 days of incubation at 20 C and is often used as a surrogate of
the degree of organic pollution in water.

Biochemical decomposition in wastewater occurs in two phases. Organic

materials are decomposed by microorganism in first phase. In second phase,
nitrification occurs where the ammonium ion is oxidized to nitrite and nitrate by
the bacteria such as nitrosomonas and nitrobacter. However, the nitrification
normally occurs few days after the BOD 5 test. Nitrification occurs faster
especially in wastewater from water treatment plant and surface water that
usually contain higher bacteria population. The occurrence could be prevented
by adding inhibitor such as N-allythiourea (C4H5N2S) into the water.

Apparatus and Equipment

BOD/Incubation bottles, 300 mL Incubator 201 C

Air pump Thermometer
Water bath 20 C Pipette, 1, 5, 10 mL
Measuring cylinder 25, 200 mL DO meter
Aspirator container Beaker 400 mL
22
Volumetric flask 1 L

Chemicals (Prepared)

1 Phosphate buffer solution: Dissolve 42.5 g of KH2PO4 in 700 mL of

distilled water. Add 8.8 g of NaOH into the solution and mix well. Add
2 g of (NH4)2SO4 into the solution and mix well. Dilute the solution to
1 L with distilled water and adjust the solution to pH 7.2. Discard the
solution if there is any sign of biological growth.

2 Magnesium sulphate solution: Dissolve 22.5 g of MgSO4.7H2O in

distilled water and dilute to 1 L.
3 Calcium chloride solution: Dissolve 27.5 g of CaCl2.6H2O in distilled
water and dilute to 1 L.
4 Ferric chloride solution: Dissolve 0.15 g of FeCl3.6H2O in distilled
water and dilute to 1 L.
5 Dilution water: Pipette 10 mL each of phosphate buffer, MgSO4,
CaCl2 and FeCl3 solutions into a 10 L aspirator container and make up
to volume, 10 L with distilled water. Saturate with dissolved oxygen
(DO) in dark by aerating with air pump overnight. Before use, bring
dilution water temperature to 20 C.
6 Glucose-glutamic acid solution (standard check solution): Separately
dry glucose and glutamic acid at 103 C for 1 hr. Add 150 mg glucose
abd 150 mg glutamic acid to distilled water and dilute to 1 L. Prepare
fresh immediately before use.

Procedure

A Determination of BOD in Sample

23
1 Bring samples to 20 C before making dilutions.
2 Prepare 4 sample dilutions in beakers as the following:

i 360 mL sample (without dilution)

ii 60 mL sample + 300 mL dilution water

iii 120 mL sample + 240 mL dilution water

iv 180 mL sample + 180 mL dilution water

3 Aerate each of the samples in step 2 for ~15 min.
4 Pour the samples into BOD bottles (overflow) and measure the initial
DO and temperature immediately after filling BOD bottles with
prepared samples before incubation. Make sure there are no bubbles
trapped in the BOD bottle during preparation. Repeat this step for
dilution water blank.
5 Stopper the BOD bottles tightly to ensure no bubbles trapped. Wrap
the bottles with aluminium foil (if necessary).
6 Incubate the bottles in the incubator 20 C for 5 days.
7 Measure final DO after 5 days incubation period.
8 Calculate the BOD5 with the following formula,

BOD5 (mg/L) = A/B (C - D)

where,

A = total volume after dilution (L)

B = sample volume used before dilution (L)

C = Total DO consumed in sample (mg/L)

D = Total DO consumed in blank (mg/L)

B Standard Check

1. Determine the BOD at 20 C for a 2% dilution of standard check

solution (7 mL of glucose-glutamic acid solution + 343 mL dilution
24
 water) in 350 mL of BOD bottle.
2. Repeat step A.4-A.8 for the standard check solution.
3. The BOD should be in the range of 19830.5 mg/L.

Data and Calculations

1. Collect all necessary data from this experiment in appropriate forms

under Results.

2. Calculate the BOD5 of samples.

3. Calculate the BOD5 of standard check solution.

Discussion guide

1. What is the rational of performing standard check?

2. What is the function of adding dilution water into sample?

3. When is the retest of sample required?

4. Discuss the results of BOD5 obtained from samples. Relate with the
pollution level.
5. Discuss the precaution steps in sampling and determination of BOD5.
6. Support your discussion with appropriate references

25
 Practical 4- Determination of Chemical Oxygen
Demand (COD)
Purpose

To determine the COD in the samples

Objectives

To introduce the method for the determination of COD

Revision/Reading

KIM3701 and KIM4701

American Public Health Association, Standard Methods for the Examination of

Water and Wastewater, 19th Edition, 1995. Chemical Oxygen Demand Closed
Reflux, Colorimetric Method, APHA 5220.D

26
Introduction

Chemical oxygen demand (COD) is a measure of the capacity of water to

consume oxygen during the decomposition of organic matter and the oxidation
of inorganic chemicals such as ammonia and nitrite. COD measurements are
commonly made on samples of waste water or of natural water contaminated by
domestic or industrial wastes. COD is measured as a standardized laboratory
assay in which a closed water sample is incubated with a strong chemical
oxidant under specific conditions of temperature and for a particular period of
time. A commonly used oxidant in COD assays is potassium dichromate
(K2Cr2O7) which is used in combination with boiling sulphuric acid (H 2SO4).
Because this chemical oxidant is not specific to oxygen-consuming chemicals
that are organic or inorganic, both of these sources of oxygen demand are
measured in a COD assay.

Chemical oxygen demand is related to biochemical oxygen demand (BOD).

However, BOD only measures the amount of oxygen consumed by microbial
oxidation and is most relevant to water rich in organic matter. It is important to
understand that COD and BOD do not necessarily measure the same types of
oxygen consumption.

The closed reflux and colorimetric method is applied in this experiment to

determine the COD in the samples. When a sample is digested, the dichromate
ion oxidizes COD material in the sample. This results in the change of
chromium from the hexavalent (VI) state to the trivalent (III) state. Both of
these chromium species are coloured and absorb in the visible region of the
spectrum.

Apparatus and Equipment

27
Digestion tubes Block heater operated at 1502 C
UV spectrophotometer Graduated pipette 5, 10 mL
Volumetric flask 1 L Beaker 2 L
Chemicals (Prepared)

1 Digestion solution, low range (for COD <100 mg/L): Dissolve 1.022 g
of K2Cr2O7 (previously dried at 150 C for 2 hrs) in 500 mL of distilled
water. Add 167 mL concentrated H2SO4 and 33.3 g HgSO4. Dissolve,
cool to room temperature, and dilute to 1 L with distilled water.
2 Sulfuric acid reagent: Add 5.5 g Ag2SO4 to 544 mL of concentrated
H2SO4. Allow to stand for 1 to 2 days to allow silver sulphate dissolve
and mix well.
3 Potassium hydrogen phthalate (KHP) standard: Lightly crush and dry
KHP to constant weight at 110 C. Dissolve 425 mg KHP in distilled
water and dilute to 1000 mL. This solution has a theoretical COD of
500 mg/L. This solution is stable when refrigerated for up to 3 months
in the absence of visible biological growth. Discard when there is
visible biological growth.
Procedure

Sampling and Storage

1. Collect samples in glass bottles. Preserve sample by acidification to

pH 2 using concentrated H2SO4.
2. Suspended matters and coloured componentes must be absent in the
sample.
Determination of COD in samples

1. Switch on block heater at least 20 min earlier to ensure temperature

reaches 150 C.
2. Transfer the following samples and solutions into separate digestion

28
 tubes :

Distilled water for Digestion

Sample
dilution solution
Blank/water 2.5 mL 0.0 mL 1.5 mL
Standard 1 0.2 mL 2.3 mL 1.5 mL
Standard 2 0.3 mL 2.2 mL 1.5 mL
Standard 3 0.4 mL 2.1 mL 1.5 mL
Standard 4 0.5 mL 2.0 mL 1.5 mL
Sample^ 0.5 mL 2.0 mL 1.5 mL
^ Calculate the Dilution Factor for sample
3. Carefully add 3.5 mL H2SO4 reagent down inside of tube so an acid
layer is formed under the sample-digestion solution layer.
4. Cap the tubes tightly. Rinse with distilled water and wipe dry. Hold the
tubes over sink and invert gently several times to mix. CAUTION:
H2SO4 acid is fuming and corrosive!
5. Place tubes on block heater at 150 C and reflux for 2 hrs. CAUTION:
Always wear face shelf and chemical resistant glove when handling hot
acid digestion.
6. Allow tubes to cool slowly to room temperature to avoid precipitate
formation. Once sample reach room temperature, loose the vial cap to
release pressure.
7. On the Window Taskbar, select Scan in Cary WinUV menu.

8. Click on Setup dialog / Scan tab to set up the Baseline and their
parameter associated with the data collection. Scan the distilled water
and save as reference/baseline value.
9.
Mix well and let suspended matter settle. Carefully remove cap to pour
to cuvette prior to UV spectrophotometer analysis.

10. Measure the digested samples, blank and standards at 420 nm

11. Calculate the COD with the following formula:

29
 mgO 2
COD as =COD calibration curve x Dilution Factor
L

Data and Calculations

1. Collect all necessary data from this experiment in appropriate forms under
Results.

2. Construct the calibration curve, concentration COD vs. Absorbance.

Absorbance data is obtain from (Blank absorbance Standard
absorbance)

3. Determine the COD of samples from above calibration curve.

Discussion guide

1. What are the main interferences in this COD test and how do the
interferences interfere the test? What is the precaution step to avoid the
interferences?

2. When is the retest of sample required?

3. What shall we do if the COD of the sample is higher than 100 mg/L?
Discuss the correct procedure to handle this high COD sample.

4. Discuss the results of COD obtained from samples. Relate with the
pollution level and BOD5.
5. Discuss the precaution steps in sampling and determination of COD.
6. Support your discussion with appropriate references.

30
 Practical 5- Determination of Ammonia in
water with Phenate Method

Purpose

To conduct ammonia analysis in water samples using colorimetric method

Objectives

To introduce the Phenate method (APHA-4500 F)

To determine the concentration of ammonia in water samples from UMT

campus

To practice data manipulation techniques including outlier removal

Revision/Reading

Standard Methods for the Examination of Water and Wastewater, 18th Edition,
AWWA, APHA, WPCF; Water Pollution Control Federation, Washington, DC,
1992.

Methods for Chemical Analysis of Water and Wastes, U.S. EPA - 600/4-79-020,
March 1979.

31
Introduction

Nitrogen is an essential element in the environment. If excess nitrogen is

present in water, however, it can result in eutrophication. Nitrogen is a nutrient
and occurs in many forms including ammonia, organic, nitrate and nitrite each
of which may be tested for in a variety of ways. Raw wastewater nitrogen is
normally present in the organic nitrogen and ammonia forms, with small
quantities of the nitrite and nitrate forms. Depending on the amount of
nitrification processes which occurs within the waste treatment plant, the
effluent may contain either ammonia or nitrate nitrogen. Under normal
circumstances, the nitrite form of nitrogen will not be present in large quantities
due to its rapid oxidation or conversion to nitrate.

The presence of large concentrations of ammonia in a stream or lake can create

a large oxygen demand. This demand is caused by the conversion of ammonia
to nitrate. High concentrations of nitrate in wastewater treatment plant effluent
can cause algae to grow in large quantities. Dead and decaying algae can cause
oxygen depletion problems which in turn can kill fish and other aquatic
organisms in streams. For this reason, testing for nitrogen in the waste water
treatment plant effluent is critical.

Apparatus and Equipment

15 ml centrifuge tube with cap Graduated pipette 10 mL

UV spectrophotometer Micropippte 1000uL
Chemicals (Prepared)

1. Stock ammonium solution (Stock A): Dissolve 1.5945 mg of anhydrous

ammonium chloride (NH4Cl), dried at 100C for 1 hour, in ammonia free
distilled water and dilute to 1000 mL.

2. Phenol reagent: dissolve add 10 g of phenol in 95% ethyl alcohol to a

final volume of 100 ml (toxic- handle with care).
32
 3. Sodium nitroprusside (nitroferricyanide): dissolve 1 g in distilled water to
a final volume of 200 ml. Store in dark bottle for not more than 1 month.
(toxic)

4. Alkaline complexing reagent: dissolve 100 g of trisodium citrate and 5 g

of sodium hydroxide in DI water to a final volume of 500 ml.

5. Sodium hypochlorite: use commercial bleach (i.e., Chlorox), as new as

possible.

6. Oxidizing solution: add 100 ml alkaline solution (4) to 25 ml sodium

hypochlorite (5). Prepare fresh daily.

7. Sample C, a quality control sample with 0.5 mg NH3-N/L.

Procedure

Preparing working standards and Sample

1. Calculate the concentration of ammoniacal-nitrogen present in Stock A

in the unit of (mg NH3-N/L); MW of anhydrous ammonium chloride
(NH4Cl) = 53.49g

2. Using 15ml centrifuge tubes, prepare the following series of working

standards. Make sure the centrifuge tubes are properly clean and 100%
dry. Use the designated pipette to transfer Stock A solution; avoid
cross sample contamination.

3. Using a clean pipette to transfer 10mL of DI water. Accuracy of

volumetric measurement is very important in standard preparation.

Theoretical
Volume of Volume of DI Concentration in
Name
Stock A (ml) water (mL) mg NH3-N/L
(fill in the blank)
Std 1 0.1 10
Std 2 0.2 10
Std 3 1.0 10
Std 4 2.0 10
Volume sample (mL)
Blank 10
Sample A 10
33
 Sample C 10
4. Calculate the theoretical concentration for each standard (1-4) in the
table.

5. To all tubes add 0.4 ml of Phenol reagent, 0.4ml nitroferricyanide and 1

ml of Oxidizing solution. Each reagent solutions has its own designated
pipette; use the right pipette to transfer reagent solutions.

6. Mix well (invert screw cap tubes) cover the sample in dark and let
colour develop at room temperature for at least 1 hour.

Setup Cary 50 for concentration determination

1. On the Window Taskbar, select Scan in Cary WinUV menu.

2. Click on Setup dialog / Scan tab to set up the Baseline and their
parameter associated with the data collection. (Ask Demonstrator).

3. Scan the deionised water spectrum and save as Baseline

4. Scan all blank, standards and samples.

5. Check all samples absorbance reading, if your sample absorbance

reading higher than Standard 4 absorbance reading, a dilution step is
required. [Dilute your samples with DI water, record the dilution factor].

6. Once all measurement has been read, copy all absorbance values into an
Excel file.

7. To save your excel data, insert a CD-R disc: click on the file menu item
and select Save Data. Select DVD RW drive (E :), enter the file name for
this Concentration run in the file name field.

8. Remark: DO NOT SAVE YOUR DATA at the PC.

Data and Calculations (Report)

1. Using Microsoft excel, construct a table for the data from UV-VIS.
2. Using the calculated Theoretical Concentration values and absorbance
data from UV-VIS, plot calibration curves for ammoniacal-nitrogen.

34
 3. Based on the obtain calibration curves, report the r2, slope and y-
intercept in a table.

Nutrient Slope y-intercept r2

NH3-N

y=mx +c ,
4. Using the formula calculate the concentration of
ammoniacal-nitrogen in all samples A, B, C. [using excel to compute
your answer, make sure your final concentration is multiply by dilution
factor if necessary].
5. Using result of sample C, calculate the percentage of recovery, %R

Obtain concentrationblamk
R= 100
Actual concentration

Questions to be answered in the report

1. For what purpose is the Sodium hypochlorite used in this experiment?

2. If ammonia testing is not going to be run immediately, how should samples

be preserved?
3. Comment on the position of your sample (A and C) NH 3-N data points in
the calibration curve does it fall within the working standard range? If
not, then can you suggest a possible reason for this? (Ensure that you
include a print out of the updated calibration plot in your report, indicate
sample A, C data point).
4. How much confidence do you have in your determination of ammonia in
water analysis? (Hint: use % recovery and R2 value to help you answer
this question)
5. The Malaysian Department of Environment (DOE) Environmental Quality
(Industrial Effluents) regulation 2009, 5th Schedule for discharge of
industrial effluent states that the ammoniacal-nitrogen concentration in the
35
 water bodies should be < 10 mg NH3-N/L. Comment on whether the
nitrogen concentration you determined might indicate a problem meeting
this statutory limit. Briefly explain your reasoning.

Practical 6- Determination of Selected Metals

in Suspended Particulate

Purpose

To conduct metal analysis in airborne particulate matter using flame AAS.

Objectives

To introduce hot acid digestion method

To learn calibration and measurement techniques for the determination of

selected metals in PM10

36
Revision/Reading

Environmental Protection Agency. 1999a. Compendium Method IO-3 2:

Determination of Metals in Ambient Particulate Matter using Atomic
Absorption (AA) Spectroscopy. EPA/625/R-96/010a.

Introduction

The air quality in Malaysia is described in terms of an Air Pollutant Index

(API). The API is an indicator of air quality and was developed based on
scientific assessments to indicate in an easily understood manner, the presence
of pollutants and their impact on health. Monitoring stations measure the
concentration of five major pollutants in the ambient air: PM10, sulfur dioxide,
nitrogen dioxide, carbon monoxide and ozone. The API index is used by
governmental agencies to characterize the quality of the ambient air at a given
location. As the AQI increases, the severity of probable adverse health effects
increases as does the percentage of the population expected to be affected by the
adverse health effects.

In Malaysia the PM10 (fine airborne particulate smaller than 10 micrometers in

diameter) comes mainly from road transportation, industrial emissions, and
open burning sources. Apart derived from anthropogenic activities, particles in
the atmosphere are also produced from natural sources such as wind-borne dust,
sea spray and volcanoes. The chemical (metals) composition of PM10 is known
to be influenced mainly by the origin of aerosol particles. Understanding the
metal contents in PM10 is hence important for the proper apportionment of
different source contributions from different pollutant emitter or sources.
Besides that, exposure to metals in the air is capable to causing negative health
effects ranging from cardiovascular and pulmonary inflammation to cancer and
damage of vital organs. Thus determination of metal concentration in PM10 can

37
provide important information on health effects with exposure to airborne
particulate matter.

Apparatus and Equipment

15 ml centrifuge tube with cap Graduated pipette 10 mL

Flame AAS 25ml volumetric glassware
100 ml glass beaker with glass watch Plastic tweezers
Zip bag DI water Wash Bottle
Chemicals (Prepared)

1. Hydrochloric Acid, concentrated 37% in 250ml reagent bottle.

2. Nitric Acid, concentrated 65% in 250ml reagent bottle.

3. Multi-element standards (50mg/L) : pipette 5ml each single-element

standards (1000mg/L) Na, Ca, Pb, Zn, Cu and Cd into a 100ml
volumetric flask and diluted to volume with 2% HNO3 acid.

Procedure

Hot acid extraction (Prepared your samples within the 1st hour of the practical)

1. PM10 sample in zip bag will be given in the size of 1x 8 strip from the
8x10 filter collected during Experiment 1.

2. A blank filter sample in the size of 1x 8 will be provided.

3. using a plastic forceps, retrieve the PM10 sample from the zip bag and the
place the filter strip into a 100ml glass beaker, place the filter strip into lower
portion of the beaker to ensure acid volume will cover the entire filter.

38
4. Repeat step 3 for blank filter sample.

5. In a fume hood, add 9 ml of concentrated Hydrochloric Acid into both PM10

and filter blank beakers. Add another 3 ml of concentrated Nitric Acid into
beakers. The acid should cover the strip completely. A full face shield and
nitrile gloves must put on all time when handling acid extraction. [Caution:
Nitric and hydrochloric acid fumes are toxic. Take extreme care not to spill
any acids in the laboratory. If spills occur, ask your lab assistants immediately
about the proper clean-up procedure].

6. Place beakers on the hot-plate, contained in a fume hood, reflux gently while
covered with a watch glass for 30 min. Do not allow sample to dry. Decrease
the heating temperature or remove the beakers from the hot-plate if necessary
to prevent sample dryness. Once complete, place remove the beakers from
hot plate and allow samples cooling in fume hood (approximately 10
minutes).

7. Rinse the beaker walls and glass wash by adding approximately 10 ml

deionized water (DI water wash bottle). Allow the samples to stand at least
15 minutes.

8. Transfer the extraction fluid (including the filter) in the beaker to a 50mL
volumetric flask. Rinse the beaker 3 times with deionized water (small
volume each time) and add the rinses to the flask. Dilute to the mark with
deionized water and shake and let the sample stand for 5 minutes.

9. Using a 15 mL centrifuge tube, carefully transfer approximately 15 ml of

sample from volumetric flask to prelabeled centrifuge tube. Make sure the
filter paper and other solid material are remaining in the flask. The clear
solution sample in centrifuges tubes is now ready for analysis.

39
Standard for Flame AAS (Prepared your standards within the first 1.5 hours of
the practical, can start the preparation simultaneously with hot acid extraction)

1. Using 15ml centrifuge tubes, prepare the following series of working

standards. Make sure the centrifuge tubes are properly clean and 100% dry. Use
the designated pipette to transfer Multi element standards solution; avoid
cross sample contamination.

2. Using a clean pipette to transfer 15mL of DI water. Accuracy of volumetric

measurement is very important in standard preparation.

3. Calculate the theoretical concentration for each standard (1-4) in the table.

volume of Theoretical
Volume of DI
Name Multi element Concentration in
water (mL)
standards (mL) mg/L (fill in the blank)
Std 1 0.5 15
Std 2 1.0 15
Std 3 1.5 15
Std 4 2 15
Setup FAAS for concentration determination

1. Set up the flame atomic absorption spectroscopy according to the

operating instructions (will be instructed by the instructor).
2. Auto zero the instrument with blank, and measure the absorbance of all
the standards and PM10 and blank filter sample.
3. Record all the data obtained (build your own table).

Data and Calculations (Report)

6. Using Microsoft excel, construct a table for the absorbance data from
FAAS.
7. Using the calculated Theoretical Concentration values and absorbance
data from FAAS, plot calibration curves for Na, Ca, Pb, Zn, Cu and Cd.
8. Based on the obtain calibration curves, report the r2, slope and y-
intercept in a table.
Metal Slope y-intercept r2

40
 Na
Ca
Pb
Zn
Cu
Cd
y=mx +c ,
4. using the formula calculate the concentration of Na, Ca, Pb,
Zn, Cu and Cd in PM10 and Blank filter in the unit of ug/m 3. [using excel
to compute your answer; Ensure that you include a print out of the
updated calibration plot in your report, indicate PM10 and filter Blank
data point).

5. Metal concentration in the air sample should be calculated as follows:

mg ( C D P )B
Metal
( )
m 3
=
A

Where,
C = concentration of metal, mg/L
D = final volume of digestion solution per filter strip, L
Total useable filter areaince s
P = proportion value of digested areaof one stripinces
B = filter blank value, mg/L
A = total air volume pull through filter in m3

Question to be answered in the report

1. Why there are negative absorbance readings and how much confidence do
you have in your determination of metals in PM10 samples using FAAS?
(Hint: use R2 value and instrument detection limit to help you answer this
question)
2. Suggest two likely sources of metal in your PM10 samples.
3. In view of your answer to No 1, suggest a possible approach to improve
detection limit of metals in PM10 using FAAS.

41
 Practical 7- Determination of Selected Ionic
Species in Particulate Matter 10m (PM10)
and rain water

Purpose

To analyze sulfate, nitrate and chloride in PM10 and rain sample

Objectives

To introduce the concentration of selected anions and cations in PM10 sample

To gain competence in the use of ion chromatography

Revision/Reading

California Environmental Protection Agency, 2015. Standard operation

procedure (SOP) for the analysis of anions and cattions in PM10 samples by ion
chromatography. www.arb.ca.gov/aaqm/sop/mld068.pdf

Introduction

Airborne particle especially fine particles are directly linked to their potential
for causing health problems. Exposure to such particles can affect both human
organs such as lungs and heart. PM10 is defined as small particles with diameter
size less than 10 micrometers and is a mixture of various substances. These
substances in PM10 occur in the form of solid particles or as liquid drops. Some
particles are emitted directly into the atmosphere. Other particles result from
gases that are transformed into particles through physical and chemical
42
processes in the atmosphere. A variety of emission sources and meteorological
processes contribute to ambient PM10.

Knowledge of the chemical substance of PM10 can indicate the source of the
PM10 and provide insight into how to control PM10. In air quality monitoring
program inorganic ion analyses of PM10 are performed to measure some of the
major secondary components of PM10. Secondary PM10 is not emitted as
particles but is formed through chemical reactions in the atmosphere. In
Malaysia, ion Analysis Chloride (Cl-), nitrate (NO3-), sulfate (SO42-), ammonium
(NH4+), and potassium (K+) are routinely measured from samples collected in
PM10 and total deposition (rain water). The acquired information is going to
assists the policy maker to design better regulatory tool to stop air pollution in
this country.

Apparatus and Equipment

50 ml centrifuge tube with cap Ion chromatography

Deionized water Wash Bottle Plastic tweezers
Ultrasonic water bath Polyethylene Sample bottles (100ml)
Dionex 10 5ml autosampler vials and Refrigerator
caps with septa
Calibrated pH meter
Chemicals (Prepared)

1. Anion and Cation Eluents

2. Anion multi-element standards (50mg/L) : pipette 5ml each single- anion

standards (Cl-), nitrate (NO3-), sulfate (SO42-), into a 100ml volumetric
flask and diluted to volume with deionised water.

3. Cation multi-element standards (50mg/L) : pipette 5ml each single-

cation standards (1000mg/L) ammonium (NH4+), and potassium (K+),

43
 sodium (Na+), calcium (Ca2+) into a 100ml volumetric flask and diluted to
volume with deionised water.

Instruction

Students are request to collect the rainwater samples at the beginning of

semester. Student can start to process the PM10 filter samples at any time from
20 March 2015 to 3 April 2015.

Procedure

PM10 Filter extraction

1. PM10 sample in zip bag will be given in the size of 1x 8 strip from the
8x10 filter collected during Experiment 1.

2. A blank filter sample in the size of 1x 8 will be provided.

3. using a plastic forceps, retrieve the PM10 sample from the zip bag and the
place the filter strip into a clean and dry 50ml centrifuge tube, place the filter
strip into lower portion of the beaker to ensure deionized water volume will
cover the entire filter.

4. Repeat step 3 for blank filter sample.

5. Dispense 25 ml of deionized water into each centrifuge tube and cap it.

6. Secure the PM10 samples and filter blank on the ultrasonic water bath and
left them for 15 minutes at medium speed.

7. After the samples have been ultrasonically extracted for 15 minutes, carefully
transfer the PM10 and filter blank extraction solution (solution only) into 5ml
autosampler tubes, wear nitrile glove when handling your samples to avoid
sample contamination.

44
8. Using marker pen (do not use labeling sticker), label the autosampler tubes as
below:

Sample Label
Tube 1 Filter Blank extraction solution Your group number - A
Tube 2 PM10 extraction solution for anion Your group number - B
Tube 3 PM10 extraction solution for cation Your group number - C
Tube 4 Rain water for anion Your group number - D
Tube 5 Rain water for cation Your group number - E

9. after extraction, bring your samples to demonstrator, the samples are stored in
a refrigerator at 4C until analysis.

Collection of rainwater

1. Build a DIY rainwater collector, cover your collector with a clean plastic bag
until deploy.

2. Deploy the DIY rain collector during raining event. Immediately after the rain
event collect the entire rain water into a pre-clean plastic bottle.

3. Record the sampling data as below

1 Sampling date in DD/MM/YY

2 Time start rain event in hh:mm
3 Time stop rain event in hh:mm
4 Volume of rainwater in ml (measure
in lab)
5 the top diameter of your rain collector
in cm
6. pH value (measure in lab)

4. Discard the samples if the rainwater volume less than 50ml. Repeat Step 2.

45
5. using a measuring cylinder measure the volume of rain water.

6. After measure the volume, carefully transfer the rainwater into 5ml
autosampler tubes, wear nitrile glove when handling your samples to avoid
sample contamination.

7. Using marker pen (do not use labeling sticker), label the autosampler tubes as
table above. Bring your samples to demonstrator; the samples are stored in a
refrigerator at 4C until analysis

8. Measure the rain water pH using the leftover sample by a calibrated pH

meter.

Data Handling

1. Cation and Anion are identified and quantified by Chromeleon Ion

Chromatography software.

2. Reports will be uploaded to e-learning after analysis run (your demonstrator

will perform the sample analysis).

3. Tabulate all of your result (including the calibration results, coefficient value
(r2), and the regression equation for each cation and anion analyte).

4. Report the concentration anion and cation of PM10 in unit of mg/m 3 (hint:
use air volume in Experiment 1 and example formula in Practical 6)

5. Report the concentration of anion and cation of Rainwater in unit of mg/L

Question

1. Explain why the level of cation and anion in both PM10 and rain water are
difference? (Hint: use sampling method and sampling strategy to explain
your answer)

2. Explain why your rain water pH value are less than 7.

46
3. When comparing with river water pH (result from Practical 1), the rainwater
pH is substantial lower. What could account for these differences? Explain.

47