ChE 3131L Laboratory Manual

Saint Louis University
SCHOOL OF ENGINEERING AND ARCHITECTURE
LABORATORY MANUAL
Experiment No: 1
Title: WATER CHARACTERIZATION AND DETERMINATION OF PHYSICAL and

CHEMICAL WATER QUALITY PARAMETERS
At the end of this experiment, the student should be able to:
TLO#2: Execute the standard operating procedures in water sampling, storage and
preservation.
TLO#3: Measure the different physical and chemical parameters of water such as pH,
TDS, temperature, turbidity and conductivity.
I. INTRODUCTION:
Water sampling and analysis involve the collection of water samples and
measurement for physical, chemical and biological characteristics to determine its quality.
These results are compared against water quality standards in regulations and guidelines
to determine its use or the treatment required to make the water suitable for its intended
use.
Water or wastewater sampling is generally performed by one of two methods, grab

sampling or composite sampling. Grab sampling is just what it sounds like; all of the test
material is collected at one time. As such, a grab sample reflects performance only at the
point in time that the sample was collected and only if the sample was properly collected.
Composite sampling consists of a collection of numerous individual discrete samples
taken at regular intervals over a period of time, usually 24 hours. The material being
sampled is collected in a common container over the sampling period. The analysis of
this material, collected over a period of time, will therefore represent the average
performance of a water or wastewater system during the collection period.
Grab sampling allows the analysis of specific types of unstable parameters such
as pH, dissolved oxygen, chlorine residual, nitrites and temperature. However, the most
widely used indicators of treatment plant performance, including CBOD5 (five day
carbonaceous biochemical oxygen demand), TSS (total suspended solids) and TN (total
nitrogen) require the use of composite sampling techniques. Standard Methods (20th
Edition, Section 1060 § B, “Collection and Sampling”) states “A sample can represent
only the composition of its source at the time and place of collection.” Grab samples may
be used to represent “some well-mixed surface waters, but rarely, wastewater streams”
for water quality evaluation. The widely varying flow patterns of residential treatment
plants make it impossible to evaluate performance by analyzing a single grab sample of
COURSE #: CHE 3131L

DESCRIPTIVE TITLE: ENVIRONMENTAL
CHEMICAL ENGINEERING DEPARTMENT
ENGINEERING FOR CHE LABORATORY
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effluent. Residential treatment plants receive a frequent number of short hydraulic surges
throughout the day followed by intermittent periods of no flow whatsoever.
Composite samples of effluent collected, stored, analyzed, tabulated and

averaged over an extended period of time provide the only verifiable indication of
treatment plant performance. Collecting and analyzing these composite samples is often
an expensive and time-consuming process. For these reasons, most regulatory
organizations recognize independent third-party certifiers, who use composite sampling
methods to conduct performance evaluation and accurately measure system
performance in a standardized, reproducible setting. Attempting to evaluate a residential
treatment system in the field by analyzing a grab sample taken from a sump or any other
containment vessel provides a compound degree of error and will yield erroneous
conclusions about system performance.
Conductivity is a measure of water’s capability to pass electrical flow. This ability

is directly related to the concentration of ions in the water. These conductive ions come
from dissolved salts and inorganic materials such as alkalis, chlorides, sulfides and
carbonate compounds. Compounds that dissolve into ions are also known as electrolytes.
The more ions that are present, the higher the conductivity of water. Likewise, the fewer
ions that are in the water, the less conductive it is. Distilled or deionized water can act as
an insulator due to its very low conductivity value. Sea water, on the other hand, has a
very high conductivity.
The pH of a solution is measured as negative logarithm of hydrogen ion

concentration. At a given temperature, the intensity of the acidic or basic character of a
solution is indicated by pH or hydrogen ion concentration. pH values from 0 to 7 are
diminishing acidic, 7 to 14 increasingly alkaline and 7 is neutral.
Turbidity can be measured by its effect on the scattering light, which is termed as
Nephelometry. Turbidimeter can be used for sample with moderate turbidity and
nephelometer for sample with low turbidity. The higher the intensity of scattered lights,
the higher the turbidity. Turbidity is an expression of the optical property that causes light
to be scattered and absorbed rather than transmitted in straight lines through the sample.
Water temperature is a physical property expressing how hot or cold water is. As
hot and cold are both arbitrary terms, temperature can further be defined as a
measurement of the average thermal energy of a substance. Temperature is also
important because of its influence on water chemistry. The rate of chemical reactions
generally increases at higher temperature. Water, particularly groundwater, with higher
temperatures can dissolve more minerals from the rocks it is in and will therefore have a
higher electrical conductivity. Warm water holds less dissolved oxygen than cool water,
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and may not contain enough dissolved oxygen for the survival of different species of
aquatic life. Some compounds are also more toxic to aquatic life at higher temperatures.
Total dissolved solids (TDS) combine the sum of all ion particles that are smaller
than 2 microns (0.0002 cm). This includes all of the disassociated electrolytes that make
up salinity concentrations, as well as other compounds such as dissolved organic matter.
In “clean” water, TDS is approximately equal to salinity. In wastewater or polluted areas,
TDS can include organic solutes in addition to the salt ions.
Physical and chemical water quality parameters need to be taken and measured
as soon as possible after sample collection thus these tests should be done on-site.
II. EQUIPMENT/ MATERIALS NEEDED:
1 TDS Meter 1 Conductivity Meter

1 pH Meter 2 Beakers
1 Colorimeter
III. PROCEDURES:
A. Water Sampling:
1. Proceed to your assigned sampling point/s.

2. Evaluate the location of your sampling point and assess all the factors that may affect
the water quality of the water body.
3. Locate the specific point where the water sample should be taken in order to obtain a
representative sample. It is done by approximating the whole area covered by the
water body including its depth and locating the center.
4. Wash the two 2-liter Absolute Distilled Drinking Water plastic bottles (water sample
container) thrice with the water sample from the exact location of the sampling point
where the sample will be taken.
5. Fill the bottles carefully with the water sample by submerging the water bottles in the
water body. Make sure that the bottles are completely filled and contain no air. As
much as possible, cover the water bottle while it is submerged in the water body.
6. Do some precautions in order not to contaminate the water sample with any
contaminants.
7. Subject the water samples taken to different water quality tests and analysis.
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B. Determination of Physical Water Quality Parameters:
a. Determination of the Temperature and pH of the Water Sample
1. Calibrate the pH meter using solutions of known pH.

2. Rinse the beaker with deionized water then with the water sample twice.
3. Place approximately 75 mL of the water sample in a 100 mL beaker.
4. Wash the electrode of the pH meter using deionized water.
5. Place (dip) the electrode of the pH meter in the water sample. Record the
temperature displayed in the pH meter.
b. Determination of the Conductivity of the Water Sample
3. Wash the electrode of the conductivity meter using deionized water.
4. Place (dip) the electrode of the conductivity meter in the water sample.
5. Record the conductivity displayed in the conductivity meter. (Note: Adjust the
setting of the conductivity meter according to the range where the conductivity of
the water sample is most probably expected in order to have an accurate reading.
This is done by shifting to the next setting from the default setting which can read
only low conductivities until a stable conductivity reading will be displayed)
c. Determination of the Turbidity of the Water Sample
1. Rinse the vial with deionized water then with water sample twice.
2. Fill the vial with water sample until it is half-filled.
3. Place the vial containing the blank solution in the colorimeter and press “zero”.
Wait until the colorimeter displays a 0 FAU reading. This is done to ensure that the
colorimeter is calibrated and ready to be used for water sample turbidity test.
4. Place the vial containing the water sample in the colorimeter and press “read”.
5. Record the turbidity displayed in the colorimeter.
6. Repeat procedure 3 before another trial or another water sample will be tested.
d. Determination of the Total Dissolved Solids (TDS) of the Water Sample
3. Wash the electrode of the TDS meter using deionized water.
4. Place (dip) the electrode of the TDS meter in the water sample while pressing the
button on the top of the TDS meter.
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5. Record the TDS displayed in the TDS meter.
IV. DATA AND RESULTS
Sample / pH Temperature Conductivity Turbidity Total

Trial Dissolved
Solids
Trial 1
Trial 2
Trial 3
V. DISCUSSION (observation/ interpretation of results):
VI. CONCLUSION (option of the department if this part will be included):
VII. REFERENCES:
Standard Methods for the Examination of Water and Wastewater

Philippine National Standards for Drinking Water – 1993
Solid Wastes Management by PMO-PTFWM, DENR-EMB
Davis, Mackenzie. Introduction to Environmental Engineering, 5th Ed. McGraw-Hill
Education, c2012.
Metcalf & Eddy. Wastewater Engineering: Treatment, Disposal and Reuse, 3rd Edition.
McGraw-Hill Education, c1991.
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Experiment No: 2
Title: DETERMINATION OF SOLIDS CONTENT OF WATER/WASTEWATER BY

GRAVIMETRIC METHOD
TLO#4: Perform the standard operating procedures in determining the amount of solids
(Total Solids, Total Suspended Solids, Total Dissolved Solids, Volatile Suspended
Solids, and Volatile Fixed Solids) present in water.
I. INTRODUCTION:
When the term “solids” in terms of water quality, two terms readily come to mind,
suspended and dissolved, of course solids are not limited to these types. There are fixed,
volatile, settleable, and total. There are many areas in which the amount of these types
of solids must be monitored, including drinking water, wastewater, industrial discharges
and process control. Suspended solids are not desirable in water used for drinking and
bathing. Dissolved solids are important to the quality of drinking water because if levels
are high, the taste of the water is affected. Solids are an important parameter to monitor
in the control of biological and physical treatment processes and for assessing
compliance with regulatory agency wastewater effluent limitations.
Solids refer to matter suspended or dissolved in water or wastewater. Solids may

affect water or effluent quality adversely in a number of ways. Waters with high dissolved
solids generally are of inferior palatability and may include an unfavourable physiological
reaction in the transient consumer. For these reasons, a limit of 500 mg dissolved solids/L
is desirable for drinking waters. Highly mineralized waters also are unsuitable for many
industrial applications. Waters high in suspended solids may be aesthetically
unsatisfactory for such purposes as bathing. Solids analyses are important in the control
of biological and physical wastewater treatment process and for assessing compliance
with regulatory agency wastewater effluent limitations.
TOTAL SOLIDS
Total Solids, TS, is a measure of all suspended, colloidal, and dissolved solids in a
sample of water. It is a term applied to the material residue left in vessel after evaporation
of a sample and its subsequent drying in an oven at a defined temperature. Total solids
include total suspended solids, the portion of total solids retained by a filter, and total
dissolved solids, the portion that passes through the filter. Total solids include dissolved
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salts such as sodium chloride, NaCl, and solid particles such as silt and plankton. An
excess of total solids in rivers and streams is a very common problem.
The term “total solids” applies to the material residue remaining in the vessel after the
evaporation and drying in an oven at 103-105C. A well mixed sample is evaporated in a
weighted dish and dried in a constant weight in an oven. The increase in weight over that
of the empty vessel represents the total solids. The total solids value includes the
combination of “total suspended solids” and “total dissolved solids”.
TOTAL SUSPENDED SOLIDS AND TOTAL DISSOLVED SOLIDS
Total suspended solids (TSS) are particles that are larger than 2 microns found in the
water column. Anything smaller than 2 microns (average filter size) is considered a
dissolved solid. Most suspended solids are made up of inorganic materials, though
bacteria and algae can also contribute to the total solids concentration. These solids
include anything drifting or floating in the water, from sediment, silt, and sand to plankton
and algae. Organic particles from decomposing materials can also contribute to the TSS
concentration. As algae, plants and animals decay, the decomposition process allows
small organic particles to break away and enter the water column as suspended solids.
Even chemical precipitates are considered a form of suspended solids. Total suspended
solids are a significant factor in observing water clarity. The more solids present in the
water, the less clear the water will be.
The “total suspended solids” portion is the solid retained on a filter of specified pore
size as a sample is drawn through the filter after drying at 103 – 105 C. A well-mixed
sample is vacuum filtered through a method specified glass-fiber filter and dried in an
oven. The filter and filter support must be prepared according to method specifications.
The increase of weight in the filter represents the suspended solids.
Total dissolved solids (TDS) combine the sum of all ion particles that are smaller than
2 microns (0.0002 cm). This includes all of the disassociated electrolytes that make up
salinity concentrations, as well as other compounds such as dissolved organic matter. In
“clean” water, TDS is approximately equal to salinity. In wastewater or polluted areas,
TDS can include organic solutes (such as hydrocarbons and urea) in addition to the salt
ions. TDS can also affect water taste, and often indicates a high alkalinity or hardness.
The “Total Dissolved Solids” are able to pass through the filter used for the total
suspended solids and are left as residue after evaporation and drying at 180 C. A well-
mixed sample is vacuum filtered through the same filter used in the total suspended solids
procedure. The filtered liquid is then evaporated in a weighed vessel on a steam stable
and then dried in an oven. The increase in the weight of the vessel represents the
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dissolved solids. The values for total suspended and total dissolved solids are affected
by the selection of filter and the preparation technique of the filtering apparatus.
TOTAL FIXED SOLIDS, TOTAL VOLATILE SOLIDS AND SETTLEABLE SOLIDS
Fixed Solids, a term applied to the residue of total, suspended, or dissolved solids
after heating to dryness for a specified time at a specified temperature. The weight loss
on ignition is called, volatile solids. Determinations of fixed and volatile solids do not
distinguish precisely between inorganic and organic matter because the loss on ignition
is not confined to organic matter. It includes losses due to decomposition or volatilization
of some mineral salts. Settleable solids are a material settling out of suspension within a
defined period. It may include floating material, depending on the technique applied.
The “Fixed Solids” is the term applied to the residue of any one of the following “Total
Solids”, “Total Suspended Solids”, or “Total Dissolved Solids” after igniting at 500
plus/minus 50oC while the weight loss after ignition is the “Volatile Solids”. If fixed and
volatile solids are being determined from the same samples being used in the
determination of total, suspended or dissolved then the drying vessels must be prepared
in accordance with instructions for fixed and volatile solids.
Volatile solids are those solids in awater or other liquids that are lost in ignition of dry
solids at 1020F (550C). It is a water quality measure obtained from the loss on ignition
of total suspended solids. It has great importance in water and wastewater treatment. It
normally represents the amount of organic solids in water. The greater the concentration
of organic or volatile solids, the stronger the wastewater is. It is helpful in assessing the
amount biologically inert organic matter, such as lignin in case of wood pulping waste
liquids.
Volatile solid is a substance that can easily transform from its solid phase to vapour
phase without going through a liquid phase. In domestic wastewater, solids are about 50
percent organic, which in turn contaminates the ground and fresh water. These solids are
generally from plants, dead animal matter, and synthetic organic compounds. They can
be ignited or burned. Because the organic fraction can be driven off at high temperatures,
they are called volatile solids.
Water which contains high levels of volatile solids is not suitable for drinking.
Settleable solids are also known as bedded sediments, or bedload. These sediments
can vary from larger sand and gravel to fine silt and clay, depending on the flow rate of
water. Sometimes these sediments can move downstream even without rejoining the
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suspended solids concentration. When settleable solids are moved along the bottom of a
body of water by a strong flow, it is called bedload transport.
The “Settleable Solids” is the term applied to the material settling out of suspension
with in a defined period of time. Settleable solids analyses are usually performed using
the volumetric or Imhoff cone and allowed to settle for 1 hour with a gentle agitation at 45
minutes. The results are read from graduations on the Imhoff cone and expressed in
millilitres per liter (mL/L).
Buchner filter Watch glass

Oven Pipet
Furnace Pipetol
Analytical balance Graduated cylinder
Crucible Wash bottle
Crucible tong
III. PROCEDURES:
a. Constant Weighing
1. Heat the crucibles and filter papers placed in a watch glass in an oven at 104oC
for an hour (Start the time at the moment when the temperature of the oven
reached 104oC).
2. Cool the crucibles and filter paper inside the oven.
3. Desiccate for 30 to 60 minutes the crucibles and filter papers once the temperature
approaches room temperature.
4. Weigh the crucibles and filter papers in an analytical balance.
5. Repeat the whole procedure until all the weights yielded a difference
approximately±0.0003 or 0 value.
6. After constant weights were achieved from the oven heating, heat the crucibles
and filter papers in a furnace at 550oC for an hour (Start the time at the moment
when the temperature of the oven reached 550oC).
7. Similarly with the oven heating, cool the crucibles and filter papers, desiccate and
weigh, until all the weights yielded a difference approximately±0.0003 or 0 value.
b. Determination of the Total Solids of Water
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1. Shake the water sample well

2. Pour 25 mL of water sample into the crucibles.
3. Heat the crucibles in the hot plate until complete evaporation of water.
4. Heat the crucibles in an oven at 104oC for an hour (Start the time at the moment
when the temperature of the oven reached 104oC).
5. Cool the crucibles, desiccate and weigh and repeat the constant weighing
procedures until all the weights yielded a difference approximately±0.0003 or 0
value.
c. Determination of the Total Suspended Solids and Total Dissolved Solids of

Water
1. Shake the water sample well.

2. Filter 25 mL of the water sample using the pre-weighed filter paper and Buchner
filter attached in a vacuum pump.
3. Transfer the filtrate into the crucibles and allow to completely evaporate in a hot
plate.
4. For TDS determination, heat the crucibles in an oven at 180oC for an hour (Start
the time at the moment when the temperature of the oven reached 180oC).
5. Cool the crucibles, desiccate and weigh and repeat the constant weighing
value.
6. For TSS determination, heat the filter paper in an oven at 104oC for an hour (Start
the time at the moment when the temperature of the oven reached 104 oC).
7. Cool the filter papers, desiccate and weigh and repeat the constant weighing
value.
d. Determination of the Total Volatile Solids and Total Fixed Solids of Water

2. After constant weights were achieved from the oven heating, heat the crucibles
and filter papers subjected in TS, TDS and TSS determination, in a furnace at
550oC for an hour (Start the time at the moment when the temperature of the oven
reached 550oC).
3. Similarly with the oven heating, cool the crucibles and filter papers, desiccate and
weigh, until all the weights yielded a difference approximately±0.0003 or 0 value.
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TS = TSS + TDS
// // //
TFS = FSS + FDS
+ + +
TVS = VSS + VDS
Sample / TS TSS TDS TFS TVS

Trial
Trial 1
Trial 2
Trial 3
Total Solids
 The amount of total solid in a water sample is computed using the formula:
(𝑨−𝑩)𝒙 𝟏𝟎𝟎𝟎
Total Solids =
𝒔𝒂𝒎𝒑𝒍𝒆 𝒗𝒐𝒍𝒖𝒎𝒆, 𝒎𝑳
Where: A = weight of dried residue + crucible, mg at 104°C oven

B = weight of crucible, mg at 550°C furnace
Total Dissolved Solids
 The amount of total dissolved solid in a water sample is computed using the formula:
(𝑨−𝑩)𝒙 𝟏𝟎𝟎𝟎
Total Dissolved Solids = 𝒔𝒂𝒎𝒑𝒍𝒆 𝒗𝒐𝒍𝒖𝒎𝒆, 𝒎𝑳
Where: A = weight of dried residue + crucible, mg at 104°C oven

B = weight of crucible, mg at 550°C furnace
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 The total dissolved solids measured using TDS meter is computed by averaging:
𝑻𝑫𝑺𝟏 + 𝑻𝑫𝑺𝟐
TDS = 𝟐
Total Volatile Solids (Dissolve and Suspended Volatile Solids)
 The amount of volatile solid in a water sample is computed using the formula:
(𝑨−𝑩)𝒙 𝟏𝟎𝟎𝟎
Volatile Solids = 𝒔𝒂𝒎𝒑𝒍𝒆 𝒗𝒐𝒍𝒖𝒎𝒆, 𝒎𝑳
Total Fixed Solids (Fixed Dissolved and Fixed Suspended Solids)
 The amount of fixed solid in a water sample is computed using the formula:
(𝑩−𝑪)𝒙 𝟏𝟎𝟎𝟎
Fixed Solids = 𝒔𝒂𝒎𝒑𝒍𝒆 𝒗𝒐𝒍𝒖𝒎𝒆, 𝒎𝑳
Where: A = weight of dried residue + crucible, mg, before ignition

B = weight of residue + crucible, mg, after ignition
C = weight of crucible, mg at 550°C furnace
 The total suspended solids in a water sample should be equal to the summation of
both FSS and VSS:
TSS = FSS + VSS
Where fixed suspended solids is computed by:

(𝑨−𝑩)𝒙 𝟏𝟎𝟎𝟎
Fixed Suspended Solids = 𝒔𝒂𝒎𝒑𝒍𝒆 𝒗𝒐𝒍𝒖𝒎𝒆, 𝒎𝑳
Where: A = weight of crucible + residue, mg, at 550°C

B = weight of crucible + residue, mg, at 550°C
C = weight of crucible + residue, mg, at 104°C
 The total dissolved solids in a water sample should be equal to the summation of both
FSS and VSS:
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TDS = FDS + VDS
VII. REFERENCES:
Standard Methods for the Examination of Water and Wastewater, 20th Edition.
CHRONHEIM, G. & W. WINK. 1942. Determination of divalent iron (by o-
nitrosophenol). Ind. Eng. Chem., Anal. Ed. 14:447.
MEHLIG, R.P. & R.H. HULETT. 1942. Spectrophotometric determination of iron
with o-phenanthroline and with nitro-o-phenanthroline. Ind. Eng. Chem., Anal.
Ed. 14:869.
CALDWELL, D.H. & R.B. ADAMS. 1946. Colorimetric determination of iron in
water with o-phenanthroline. J. Amer. Water Works Assoc. 38: 727.
WELCHER, F.J. 1947. Organic Analytical Reagents. D. Van Nostrand Co., Princeton,
N.J., Vol. 3, pp. 85–93.
KOLTHOFF, I.M., T.S. LEE & D.L. LEUSSING. 1948. Equilibrium and kinetic
studies on the formation and dissociation of ferroin and ferrin. Anal. Chem.
20:985.
RYAN, J.A. & G.H. BOTHAM. 1949. Iron in aluminum alloys: Colorimetric
determination using 1,10-phenanthroline. Anal. Chem. 21:1521.
REITZ, L.K., A.S. O’BRIEN & T.L. DAVIS. 1950. Evaluation of three iron methods using
a factorial experiment. Anal. Chem. 22:1470.
SANDELL, E.B. 1959. Chapter 22 in Colorimetric Determination of Traces of Metals,
3rd ed. Interscience Publishers, New York, N.Y.
SKOUGSTAD, M.W., M.J. FISHMAN, L.C. FRIEDMAN, D.E. ERDMANN & S.S.
DUNCAN. 1979. Methods for Determination of Inorganic Substances in Water and
Fluvial Sediment. Chapter A1 in Book 5, Techniques of Water Resources
Investigations of the United States Geological Survey. U.S. Geological Surv.,
Washington, D.C.
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Experiment No: 3
Title: DETERMINATION OF CHLORIDES CONTENT OF WATER/WASTEWATER BY

ARGENTOMETRIC METHOD
TLO#5: Demonstrate how chlorides content of water is determined.
I. INTRODUCTION:
Chloride, in the form of chloride (Cl–) ion, is one of the major inorganic anions in water
and wastewater. The salty taste produced by chloride concentrations is variable and
dependent on the chemical composition of water. Some waters containing 250 mg Cl –/L
may have a detectable salty taste if the cation is sodium. On the other hand, the typical
salty taste may be absent in waters containing as much as 1000 mg/L when the
predominant cations are calcium and magnesium.
The chloride concentration is higher in wastewater than in raw water because sodium
chloride (NaCl) is a common article of diet and passes unchanged through the digestive
system. Along the sea coast, chloride may be present in high concentrations because of
leakage of salt water into the sewerage system. It also may be increased by industrial
processes.
High chloride content may harm metallic pipes and structures, as well as growing
plants.
The method used in the determination of the chloride content of the water sample is
the Argentometric Method or Mohr Method. The Mohr method of determination of
chlorides by titration with silver nitrate is one of the oldest titration methods still in use - it
was researched and published by Karl Friedrich Mohr in 1856.
Argentometric Method
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In a neutral or slightly alkaline solution, potassium chromate can indicate the end
point of the silver nitrate titration of chloride. Silver chloride is precipitated quantitatively
before red silver chromate is formed.
Erlenmeyer flask Buret

Pipet Buret clamp
Pipetol Iron Stand
Graduated cylinder Wash bottle
III. PROCEDURES:
REAGENTS
1. Potassium chromate indicator solution:
Dissolve 50 g K2CrO4 in a little distilled water. Add AgNO3 solution until a definite
red precipitate is formed. Let it stand for 12 h, filter, and dilute to 1 L with distilled
water.
2. Standard silver nitrate titrant, 0.0141M (0.0141N):
Dissolve 2.395 g AgNO3 in distilled water and dilute to 1000 mL.

Standardize against NaCl. Store in a brown bottle.1.00 mL = 500 µg Cl–
3. Standard sodium chloride, 0.0141M (0.0141N):
Dissolve 824.0 mg NaCl (dried at 140°C) in distilled water and dilute to 1000
mL; 1.00 mL = 500 µg Cl–.
a. Standardization of Silver Nitrate Titrant Against Standard Sodium Chloride
1. Pour 10 mL standard sodium chloride solution into an Erlenmeyer flask.

2. Add 1 mL potassium chromate indicator.
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3. Titrate the solution with silver nitrate titrant slowly, with continuous swirling, until a
pinkish yellow endpoint appears. Be consistent in end-point recognition.
4. Repeat all procedures for two more trials.
b. Determination of the Chloride Content of Water

2. Check the pH of the sample if it is within the pH range of 7 to 10. Adjust sample
pH to 7 to 10 with H2SO4 or NaOH if it is not in this range.
3. Place 100 mL water sample in an Erlenmeyer flask.
4. Add 1 mL potassium chromate indicator.
5. Titrate the solution with silver nitrate titrant slowly, with continuous swirling, until a
pinkish yellow endpoint appears. Be consistent in end-point recognition.
Sample / mL AgNO3 mg/L Cl- mg/L NaCl

Trial used
Trial 1
Trial 2
Trial 3
The standardized concentration of the standard silver nitrate solution was

computed utilizing the dilution formula:
𝑪 𝟏 𝑽𝟏 = 𝑪 𝟐 𝑽 𝟐 (1)
𝒎𝒈 (𝒎𝑳 𝒕𝒊𝒕𝒓𝒂𝒕𝒊𝒐𝒏 𝒇𝒐𝒓 𝒔𝒂𝒎𝒑𝒍𝒆) 𝑿 (𝒏𝒐𝒓𝒎𝒂𝒍𝒊𝒕𝒚 𝒐𝒇 𝑨𝒈𝑵𝑶𝟑)𝑿 𝟑𝟓, 𝟒𝟓𝟎

𝑪𝒍 = (𝟐)
𝑳 𝒎𝑳 𝒔𝒂𝒎𝒑𝒍𝒆
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LABORATORY MANUAL
𝒎𝒈 𝒎𝒈
𝑵𝒂𝑪𝒍 = 𝑪𝒍 𝒙 𝟏. 𝟔𝟓 (𝟑)
𝑳 𝑳
VII. REFERENCES:
HAZEN, A. 1889. On the determination of chlorine in water. Amer. Chem. J. 11:409.
KOLTHOFF, I.M. & V.A. STENGER. 1947. Volumetric Analysis, 2nd ed. Vol. 2.
Interscience Publishers, New York, N.Y., pp. 242–245, 256–258.
PAUSTIAN, P. 1987. A novel method to calculate the Mohr chloride titration. In
Advances in Water Analysis and Treatment, Proc. 14th Annu. AWWA Water
Quality Technology Conf., November 16-20, 1986, Portland, Ore., p. 673.
American Water Works Assoc., Denver, Colo.
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LABORATORY MANUAL
Experiment No: 4
Title: DETERMINATION OF IRON CONTENT OF WATER/WASTEWATER BY

POTASSIUM PERMANGANATE TITRIMETRIC METHOD
TLO#5: Demonstrate how iron content of water is determined.
I. INTRODUCTION:
Iron (Fe) is the first element in Group VIII of the periodic table; it has an atomic number
of 26, an atomic weight of 55.85, and common valences of 2 and 3 (and occasionally
valences of 1, 4, and 6). The average abundance of Fe in the earth’s crust is 6.22%; in
soils Fe ranges from 0.5 to 4.3%; in streams it averages about 0.7 mg/L; and in
groundwater it is 0.1 to 10 mg/L. Iron occurs in the minerals hematite, magnetite, taconite,
and pyrite. It is widely used in steel and in other alloys.
The solubility of ferrous ion (Fe2+) is controlled by the carbonate concentration.

Because groundwater is often anoxic, any soluble iron in groundwater is usually in the
ferrous state. On exposure to air or addition of oxidants, ferrous iron is oxidized to the
ferric state (Fe3+) and may hydrolyze to form red, insoluble hydrated ferric oxide. In the
absence of complex-forming ions, ferric iron is not significantly soluble unless the pH is
very low.
Elevated iron levels in water can cause stains in plumbing, laundry, and cooking
utensils, and can impart objectionable tastes and colors to foods. The United Nations
Food and Agriculture Organization recommended level for irrigation waters is 5 mg/L. The
U.S. EPA secondary drinking water standard MCL is 0.3 mg/L.
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LABORATORY MANUAL
Erlenmeyer flask Buret clamp

Pipet Iron Stand
Pipetol Iron ring
Graduated cylinder Wire gauze
Buret Wash bottle
III. PROCEDURES:
REAGENTS
1. Sulfuric Acid, 3 M:
Dilute sufficient amount of concentrated laboratory grade H2SO4 to prepare

a desired volume of 3 M H2SO4.
2. Potassium Permanganate, 0.1 M:
Dissolve 3.95 g of KMnO4 in sufficient amount of water in a beaker and

transfer to 250 mL volumetric flask and dilute up to the mark.
3. Standard Oxalic Acid Solution:
Weigh 5 g of oxalic acid (H2C2O4·2H2O), dissolve in sufficient amount of

water in a beaker and transfer to 250 mL volumetric flask and dilute up to the mark.
a. Standardization of KMnO4 Titrant Against Standard Oxalic Acid Solution
1. Pour 10 mL standard oxalic acid solution into an Erlenmeyer flask.

2. Add 8 mL 3 M H2SO4
3. Titrate the solution with KMnO4 titrant slowly, with continuous swirling, until a light
pink endpoint appears.
b. Determination of the Iron Content of Water
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LABORATORY MANUAL

3. Add 8 mL 3 M H2SO4
4. Titrate the solution with standard KMnO4 titrant slowly, with continuous swirling,
until a light pink endpoint appears.
Sample / mL KMnO4 mg/L Fe

Trial used
Trial 1
Trial 2
Trial 3
The standardized concentration of the standard KMnO 4 solution was computed

utilizing the formula:
mmol KMnO4 = mmol H2C2O4·2H2O (1)
which may be expanded and manipulated to calculate for the concentration of KMnO4
as,
(m H2C2O4·2H2O) (V H2C2O4·2H2O)
C KMnO4 = (2)
(MW H2C2O4·2H2O) (Vsolution)(VKMnO4)
where C represents the concentration in molarity, m the mass of oxalic acid used in
grams, MW the molecular weight in g/mol, and V the volume in liters for the solution and
in milliliters for KMnO4 and H2C2O4·2H2O.
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The average standard concentration of KMnO4 is to be used in calculating the

concentration of iron (II) in terms of mg/L by,
𝑚𝑔 (f KMnO4)(𝑉 𝐾𝑀𝑛𝑂4)(𝐶 𝐾𝑀𝑛𝑂4,𝑎𝑣𝑒)(MW Fe2+)(1000)

𝐹𝑒2+= (f Fe2+)(𝑉𝑠𝑎𝑚𝑝𝑙𝑒)
(3)
𝐿
where f stands for the factors KMnO4 and iron (II) respectively, known from the reduction-
oxidation reaction of iron and potassium permanganate. The volumes V in mL correspond
to the volume used for titration and C to the average standard concentration of KMnO4.
VII. REFERENCES:
CHRONHEIM, G. & W. WINK. 1942. Determination of divalent iron (by o-
nitrosophenol). Ind. Eng. Chem., Anal. Ed. 14:447.
MEHLIG, R.P. & R.H. HULETT. 1942. Spectrophotometric determination of iron
with o-phenanthroline and with nitro-o-phenanthroline. Ind. Eng. Chem., Anal.
Ed. 14:869.
CALDWELL, D.H. & R.B. ADAMS. 1946. Colorimetric determination of iron in
water with o-phenanthroline. J. Amer. Water Works Assoc. 38: 727.
WELCHER, F.J. 1947. Organic Analytical Reagents. D. Van Nostrand Co., Princeton,
N.J., Vol. 3, pp. 85–93.
KOLTHOFF, I.M., T.S. LEE & D.L. LEUSSING. 1948. Equilibrium and kinetic
studies on the formation and dissociation of ferroin and ferrin. Anal. Chem.
20:985.
RYAN, J.A. & G.H. BOTHAM. 1949. Iron in aluminum alloys: Colorimetric
determination using 1,10-phenanthroline. Anal. Chem. 21:1521.
REITZ, L.K., A.S. O’BRIEN & T.L. DAVIS. 1950. Evaluation of three iron methods using
a factorial experiment. Anal. Chem. 22:1470.
SANDELL, E.B. 1959. Chapter 22 in Colorimetric Determination of Traces of Metals,
3rd ed. Interscience Publishers, New York, N.Y.
SKOUGSTAD, M.W., M.J. FISHMAN, L.C. FRIEDMAN, D.E. ERDMANN & S.S.
DUNCAN. 1979. Methods for Determination of Inorganic Substances in Water and
Fluvial Sediment. Chapter A1 in Book 5, Techniques of Water Resources
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Investigations of the United States Geological Survey. U.S. Geological Surv.,

Washington, D.C.
Experiment No: 5
Title: DETERMINATION OF WATER/WASTEWATER HARDNESS BY EDTA

TITRIMETRIC METHOD
TLO#6: Execute the procedures in determining the hardness of water and infer whether
the water is soft, moderately hard, hard or very hard.
I. INTRODUCTION:
Originally, water hardness was understood to be a measure of the capacity of water

to precipitate soap. Soap is precipitated chiefly by the calcium and magnesium ions
present. Other polyvalent cations also may precipitate soap, but they often are in complex
forms, frequently with organic constituents, and their role in water hardness may be
minimal and difficult to define. In conformity with current practice, total hardness is defined
as the sum of the calcium and magnesium concentrations, both expressed as calcium
carbonate, in milligrams per liter. Water with high hardness values are referred to as
"hard," while those with low hardness values are "soft".
When hardness numerically is greater than the sum of carbonate and bicarbonate
alkalinity, that amount of hardness equivalent to the total alkalinity is called ‘‘carbonate
hardness’’; the amount of hardness in excess of this is called ‘‘noncarbonate hardness.’’
When the hardness numerically is equal to or less than the sum of carbonate and
bicarbonate alkalinity, all hardness is carbonate hardness and noncarbonate hardness is
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LABORATORY MANUAL
absent. The hardness may range from zero to hundreds of milligrams per liter, depending
on the source and treatment to which the water has been subjected.
Table 1.1: Classes of Water Hardness Based on Hardness Range
Hardness Range Hardness

(mg/L as CaCO3) Description
0 - 50 Soft
151 – 300 Hard
>300 Very Hard
EDTA Titrimetric Method
Ethylenediaminetetraacetic acid and its sodium salts (abbreviated EDTA) form a

chelated soluble complex when added to a solution of certain metal cations. If a small
amount of a dye such as Eriochrome Black T or Calmagite is added to an aqueous
solution containing calcium and magnesium ions at a pH of 10.0 ± 0.1, the solution
becomes wine red. If EDTA is added as a titrant, the calcium and magnesium will be
complexed, and when all of the magnesium and calcium has been complexed the solution
turns from wine red to blue, marking the end point of the titration. Magnesium ion must
be present to yield a satisfactory end point. To insure this, a small amount of
complexometrically neutral magnesium salt of EDTA is added to the buffer; this
automatically introduces sufficient magnesium and obviates the need for a blank
correction.
The sharpness of the end point increases with increasing pH. However, the pH cannot
be increased indefinitely because of the danger of precipitating calcium carbonate,
CaCO3, or magnesium hydroxide, Mg(OH)2, and because the dye changes color at high
pH values. The specified pH of 10.0 ± 0.1 is a satisfactory compromise. A limit of 5 min is
set for the duration of the titration to minimize the tendency toward CaCO 3 precipitation.
Erlenmeyer flask Pipetol

Pipet Graduated cylinder
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Buret Wire gauze

Buret clamp Bunsen burner
Iron Stand Wash bottle
Iron ring
III. PROCEDURES:
REAGENTS
1. Buffer solution
Dissolve 16.9 g ammonium chloride (NH4Cl) in 143 mL conc ammonium

hydroxide (NH4OH). Add 1.25 g magnesium salt of EDTA (available commercially)
and dilute to 250 mL with distilled water.
If the magnesium salt of EDTA is unavailable, dissolve 1.179 g disodium
salt of ethylenediaminetetraacetic acid dihydrate (analytical reagent grade) and
780 mg magnesium sulfate (MgSO 4⋅7H2O) or 644 mg magnesium chloride
(MgCl2⋅6H2O) in 50 mL distilled water.
Add this solution to 16.9 g NH4Cl and 143 mL conc NH4OH with mixing and
dilute to 250 mL with distilled water. To attain the highest accuracy, adjust to exact
equivalence through appropriate addition of a small amount of EDTA or MgSO 4 or
MgCl2. Store Solution 1) or 2) in a plastic or borosilicate glass container for no
longer than 1 month. Stopper tightly to prevent loss of ammonia (NH3) or pickup of
carbon dioxide (CO2). Dispense buffer solution by means of a bulb-operated pipet.
Discard buffer when 1 or 2 mL added to the sample fails to produce a pH of 10.0
± 0.1 at the titration end point.
2. Eriochrome Black T:
Sodium salt of 1-(1-hydroxy-2-naphthylazo)-5-nitro-2-naphthol-4-sulfonic

acid; No. 203 in the Color Index. Dissolve 0.5 g dye in 100 g 2,2′,2′′-nitrilotriethanol
(also called triethanolamine) or 2-methoxymethanol (also called ethylene glycol
monomethyl ether). Add 2 drops per 50 mL solution to be titrated. Adjust volume if
necessary.
3. Standard EDTA titrant, 0.01M:
Weigh 3.723 g analytical reagent-grade disodium

ethylenediaminetetraacetate dihydrate, also called (ethylenedinitrilo)tetraacetic
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LABORATORY MANUAL
acid disodium salt (EDTA), dissolve in distilled water, and dilute to 1000 mL.
Standardize against standard calcium solution. Because the titrant extracts
hardness-producing cations from soft-glass containers, store in polyethylene
(preferable) or borosilicate glass bottles. Compensate for gradual deterioration by
periodic restandardization and by using a suitable correction factor.
4. Standard calcium solution:
Weigh 1.000 g anhydrous CaCO3 powder (primary standard or special

reagent low in heavy metals, alkalis, and magnesium) into a 500-mL Erlenmeyer
flask. Place a funnel in the flask neck and add, a little at a time, 1 + 1 HCl until all
CaCO3 has dissolved. Add 200 mL distilled water and boil for a few minutes to
expel CO2. Cool, add a few drops of methyl red indicator, and adjust to the
intermediate orange color by adding 3N NH4OH or 1 + 1 HCl, as required. Transfer
quantitatively and dilute to 1000 mL with distilled water. (Note: 1 mL = 1.00 mg
CaCO3)
5. Sodium hydroxide, NaOH, 0.1N.

a. Standardization of EDTA Titrant Against Standard Calcium Solution
1. Place 10 mL standard calcium solution in an Erlenmeyer flask.

2. Add 1 to 2 mL buffer solution.
3. Add 1 to 2 drops of EBT indicator.
4. Titrate the sample with EDTA titrant slowly, with continuous stirring, until the last
reddish tinge disappears. Add the last few drops at 3- to 5-s intervals. At the end
point the solution normally is blue.
b. Determination of the Total Hardness of Water

3. Add 1 to 2 mL buffer solution. Usually 1 mL will be sufficient to give a pH of 10.0
to 10.1. The absence of a sharp end-point color change in the titration usually
means that an inhibitor must be added at this point or that the indicator has
deteriorated.
5. Titrate the sample with standard EDTA titrant slowly, with continuous stirring, until
the last reddish tinge disappears. Add the last few drops at 3- to 5-s intervals. At
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LABORATORY MANUAL
the end point the solution normally is blue. Daylight or a daylight fluorescent lamp
is recommended highly because ordinary incandescent lights tend to produce a
reddish tinge in the blue at the end point.
c. Determination of the Permanent Hardness of Water
1. Boil around 250 mL water sample for 15 minutes.

2. Cool the water sample to room temperature.
4. Add 1 to 2 mL buffer solution. Usually 1 mL will be sufficient to give a pH of 10.0
to 10.1. The absence of a sharp end-point color change in the titration usually
means that an inhibitor must be added at this point or that the indicator has
deteriorated.
6. Titrate the sample with standard EDTA titrant slowly, with continuous stirring, until
the last reddish tinge disappears. Add the last few drops at 3- to 5-s intervals. At
the end point the solution normally is blue. Daylight or a daylight fluorescent lamp
is recommended highly because ordinary incandescent lights tend to produce a
reddish tinge in the blue at the end point.
7. Repeat procedures 3 - 6 for two more trials.
Sample / mL EDTA Total mL EDTA Permanent Temporary

Trial used for Solids, used for Hardness,
Total mg/L Permanent Hardness,
Hardness CaCO3 mg/L as
Hardness CaCO3 mg/L as
CaCO3
Trial 1
Trial 2
Trial 3
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LABORATORY MANUAL
The standardized concentration of the standard EDTA solution was computed

utilizing the dilution formula:
𝑪 𝟏 𝑽𝟏 = 𝑪 𝟐 𝑽𝟐 (1)
Total Hardness = Permanent Hardness + Non-Permanent Hardness (2)
𝑨× 𝑩×𝟏𝟎𝟎𝟎
𝒎𝒈/𝑳 𝑪𝒂𝑪𝑶𝟑 = (3)
𝒎𝒍 𝒔𝒂𝒎𝒑𝒍𝒆
Where A is mL EDTA used during titration and B is the mg CaCO 3 equivalent to

1.00 mL EDTA titrant.
VII. REFERENCES:
CONNORS, J.J. 1950. Advances in chemical and colorimetric methods. J. Amer.
Water Works Assoc. 42:33.
DIEHL, H., C.A. GOETZ & C.C. HACH. 1950. The versenate titration for total
hardness. J. Amer. Water Works Assoc. 42:40.
BETZ, J.D. & C.A. NOLL. 1950. Total hardness determination by direct colorimetric
titration. J. Amer. Water Works Assoc. 42:49.
GOETZ, C.A., T.C. LOOMIS & H. DIEHL. 1950. Total hardness in water: The stability
of standard disodium dihydrogen ethylenediaminetetraacetate solutions. Anal.
Chem. 22:798.
DISKANT, E.M. 1952. Stable indicator solutions for complexometric determination
of total hardness in water. Anal. Chem. 24:1856.
BARNARD, A.J., JR., W.C. BROAD & H. FLASCHKA. 1956 & 1957. The EDTA
titration. Chemist Analyst 45:86 & 46:46.
GOETZ, C.A. & R.C. SMITH. 1959. Evaluation of various methods and reagents for
total hardness and calcium hardness in water. Iowa State J. Sci. 34:81 (Aug. 15).
SCHWARZENBACH, G. & H. FLASCHKA. 1969. Complexometric Titrations, 2nd
ed. Barnes & Noble, Inc., New York, N.Y.
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Davis, Mackenzie. Introduction to Environmental Engineering, 5th Ed. McGraw-Hill

Education, c2012.
Metcalf & Eddy. Wastewater Engineering: Treatment, Disposal and Reuse, 3 rd Edition.
McGraw-Hill Education, c1991.
Experiment No: 6
Title: DETERMINATION OF NITRATES CONTENT OF WATER/WASTEWATER BY

ULTRAVIOLET SPECTROPHOTOMETRIC SCREENING METHOD
TLO#7: Measure the amount of Nitrates level in water.
I. INTRODUCTION:
In waters and wastewaters the forms of nitrogen of greatest interest are, in order of
decreasing oxidation state, nitrate, nitrite, ammonia, and organic nitrogen. All these
forms of nitrogen, as well as nitrogen gas (N2), are biochemically interconvertible and
are components of the nitrogen cycle. They are of interest for many reasons.
Determination of nitrate (NO3–) is difficult because of the relatively complex

procedures required, the high probability that interfering constituents will be present, and
the limited concentration ranges of the various techniques.
An ultraviolet (UV) technique that measures the absorbance of NO 3– at 220 nm is

suitable for screening uncontaminated water (low in organic matter).
Ultraviolet Spectrophotometric Screening Method
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The NO3– calibration curve follows Beer’s law up to 11 mg N/L.
Measurement of UV absorption at 220 nm enables rapid determination of NO 3–.

Because dissolved organic matter also may absorb at 220 nm and NO 3– does not
absorb at 275 nm, a second measurement made at 275 nm may be used to correct the
NO3– value. The extent of this empirical correction is related to the nature and
concentration of organic matter and may vary from one water to another. Consequently,
this method is not recommended if a significant correction for organic matter
absorbance is required, although it may be useful in monitoring NO3– levels within a
water body with a constant type of organic matter. Correction factors for organic matter
absorbance can be established by the method of additions in combination with analysis
of the original NO3– content by another method. Sample filtration is intended to remove
possible interference from suspended particles. Acidification with 1N HCl is designed to
prevent interference from hydroxide or carbonate concentrations up to 1000 mg
CaCO3/L. Chloride has no effect on the determination.
Spectrophotometer Graduated cylinder

Erlenmeyer flask Buret
Pipet Wash bottle
Pipetol
III. PROCEDURES:
REAGENTS
1. Nitrate-free water:
Use redistilled or distilled, deionized water of highest purity to prepare all

solutions and dilutions.
2. Stock nitrate solution:
Dry potassium nitrate (KNO3) in an oven at 105°C for 24 h.

Dissolve 0.7218 g in water and dilute to 1000 mL. Preserve with 2 mL CHCl 3/L.
This solution is stable for at least 6 months. 1.00 mL = 100 µg NO3–-N
3. Intermediate nitrate solution:
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Dilute 100 mL stock nitrate solution to 1000 mL with water. Preserve with 2
mL CHCl3/L. This solution is stable for 6 months. 1.00 mL = 10.0 µg NO3–-N
Hydrochloric acid solution, HCl, 1N:
Dilute sufficient amount of concentrated laboratory grade HCl to prepare a

desired volume of 1 N HCl.
a. Treatment of the Sample

2. Pour 50 mL of water sample into an Erlenmeyer flask.
3. Add 1 mL HCl solution and mix thoroughly.
4. Read absorbance or transmittance against redistilled water set at zero absorbance
or 100% transmittance. Use a wavelength of 220 nm to obtain NO3– reading and a
wavelength of 275 nm to determine interference due to dissolved organic matter.
b. Preparation of Standard Curve
1. Prepare NO3– calibration standards in the range 0 to 7 mg NO3–-N/L by diluting to

50 mL the following volumes of intermediate nitrate solution: 0, 1.00, 2.00, 4.00,
7.00 . . . 35.0 mL. Treat NO3– standards in same manner as samples.
Sample / Absorbance mg/L NO3-

Trial
Trial 1
Trial 2
Trial 3
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For samples and standards, subtract two times the absorbance reading at 275 nm
from the reading at 220 nm to obtain absorbance due to NO 3–. Construct a standard
curve by plotting absorbance due to NO3– against NO3–-N concentration of standard.
Using corrected sample absorbances, obtain sample concentrations directly from
standard curve. NOTE: If correction value is more than 10% of the reading at 220 nm,
do not use this method.
VII. REFERENCES:
HOATHER, R.C. & R.F. RACKMAN. 1959. Oxidized nitrogen and sewage effluents
observed by ultraviolet spectrophotometry. Analyst 84:549. GOLDMAN, E. & R.
JACOBS. 1961. Determination of nitrates by ultraviolet absorption. J. Amer. Water
Works Assoc. 53:187.
ARMSTRONG, F.A.J. 1963. Determination of nitrate in water by ultraviolet
spectrophotometry. Anal. Chem. 35:1292.
NAVONE, R. 1964. Proposed method for nitrate in potable waters. J. Amer. Water
Works Assoc. 56:781.
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Experiment No: 7
Title: DETERMINATION OF PHOSPHATES CONTENT OF WATER/WASTEWATER

BY ASCORBIC ACID METHOD
TLO#8: Execute the standard procedures in determining Phosphates content of water.
I. 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.
These forms of phosphate arise from a variety of sources. Small amounts of

orthophosphate or certain condensed phosphates are added to some water supplies
during treatment. Larger quantities of the same compounds may be added when the
water is used for laundering or other cleaning, because these materials are major
constituents of many commercial cleaning preparations. Phosphates are used
extensively in the treatment of boiler waters. Orthophosphates applied to agricultural or
residential cultivated land as fertilizers are carried into surface waters with storm runoff
and to a lesser extent with melting snow. Organic phosphates are formed primarily by
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LABORATORY MANUAL
biological processes. They are contributed to sewage by body wastes and food residues,
and also may be formed from orthophosphates in biological treatment processes or by
receiving water biota.
Phosphorus is essential to the growth of organisms and can be the nutrient that limits
the primary productivity of a body of water. In instances where phosphate is a growth-
limiting nutrient, the discharge of raw or treated wastewater, agricultural drainage, or
certain industrial wastes to that water may stimulate the growth of photosynthetic aquatic
micro- and macroorganisms in nuisance quantities.
Phosphates also occur in bottom sediments and in biological sludges, both as

precipitated inorganic forms and incorporated into organic compounds.
Ascorbic Acid Method
Ammonium molybdate and potassium antimonyl tartrate react in acid medium with
orthophosphate to form a heteropoly acid—phosphomolybdic acid—that is reduced to
intensely colored molybdenum blue by ascorbic acid.
Spectrophotometer Pipetol
Erlenmeyer flask Graduated cylinder
Pipet Wash bottle
III. PROCEDURES:
REAGENTS
1. Sulfuric acid, H2SO4, 5N:

a desired volume of 5 N H2SO4.
2. Potassium antimonyl tartrate solution:
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Dissolve 1.3715 g K(SbO)C4H4O6⋅1/2H2O in 400 mL distilled water in a 500-

mL volumetric flask and dilute to volume. Store it in a glass-stoppered bottle.
3. Ammonium molybdate solution:
Dissolve 20 g (NH4)6Mo7O24⋅ 4H2O in 500 mL distilled water. Store it in a

glass-stoppered bottle.
Ascorbic acid, 0.1M:
Dissolve 1.76 g ascorbic acid in 100 mL distilled water. The solution is

stable for about 1 week at 4°C.
Combined reagent:
Mix the above reagents in the following proportions for 100 mL of the
combined reagent: 50 mL 5N H2SO4, 5 mL potassium antimonyl tartrate solution,
15 mL ammonium molybdate solution, and 30 mL ascorbic acid solution. Mix after
addition of each reagent. Let all reagents reach room temperature before they are
mixed and mix in the order given. If turbidity forms in the combined reagent, shake
and let stand for a few minutes until turbidity disappears before proceeding. The
reagent is stable for 4 h.
6. Stock phosphate solution:
Dissolve in distilled water 219.5 mg anhydrous KH2PO4 and dilute to 1000

mL; 1.00 mL = 50.0 µg PO43–-P.
7. Standard phosphate solution:
Dilute 50.0 mL stock phosphate solution to 1000 mL with distilled water;

1.00 mL = 2.50 µg P.

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3. Add 1 drop phenolphthalein indicator. If a red color develops add 5 N H2SO4

solution dropwise to just discharge the color.
4. Add 8.0 mL combined reagent and mix thoroughly.
5. After at least 10 min but no more than 30 min, measure absorbance of each sample
at 880 nm, using reagent blank as the reference solution.
1. Prepare individual calibration curves from a series of six standards within the
phosphate ranges. Use a distilled water blank with the combined reagent to make
photometric readings for the calibration curve.
Note: All solutions or samples containing combined reagent must be poured into a
waste bottle after the experiment.
Sample / Absorbance mg/L PO4-

Trial
Trial 1
Trial 2
Trial 3
Plot absorbance vs. phosphate concentration to give a straight line passing

through the origin and test at least one phosphate standard with each set of samples.
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VII. REFERENCES:
EDWARDS, G.P., A.H. MOLOF & R.W. SCHNEEMAN. 1965. Determination of
orthophosphate in fresh and saline waters. J. Amer. Water Works Assoc. 57:917.
MURPHY, J. & J. RILEY. 1962. A modified single solution method for the determination
of phosphate in natural waters. Anal. Chim. Acta 27:31.
SLETTEN, O. & C.M. BACH. 1961. Modified stannous chloride reagent for
orthophosphate determination. J. Amer. Water Works Assoc. 53: 1031.
STRICKLAND, J.D.H. & T.R. PARSONS. 1965. A Manual of Sea Water Analysis, 2nd
ed. Fisheries Research Board of Canada, Ottawa.
Experiment No: 8
Title: DETERMINATION OF AMMONIA CONTENT OF WATER/WASTEWATER BY

PHENATE METHOD
TLO#7: Measure the amount of Ammonia level in water.
I. INTRODUCTION:
Ammonia is present naturally in surface and wastewaters, though in very small

amounts, as a result of microbiological activity which causes the reduction of nitrogen-
containing compounds. When present in levels above 0.1 mg/l N, sewage or industrial
contamination may be indicated.
Its concentration generally is low in groundwaters because it adsorbs to soil particles

and clays and is not leached readily from soils. It is produced largely by deamination of
organic nitrogen-containing compounds and by hydrolysis of urea. At some water
treatment plants ammonia is added to react with chlorine to form combined chlorine
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residual. Ammonia concentrations encountered in water vary from less than 10 μg

ammonia nitrogen/L in some natural surface and groundwaters to more than 30 mg/L in
some wastewaters.
From the viewpoint of human health the significance of ammonia is marked because
it indicates the possibility of sewage pollution and the consequent possible presence of
pathogenic microorganisms.
The two major factors that influence selection of the method to determine ammonia
are concentration and presence of interferences. In general, direct manual determination
of low concentrations of ammonia is confined to drinking waters, clean surface or
groundwater, and good-quality nitrified wastewater effluent. In other instances, and where
interferences are present or greater precision is necessary, a preliminary distillation step
is required.
Phenate Method
The manual phenate method is applicable to both fresh water and seawater and is
linear to 0.6 mg NH3-N/L. Distill into sulfuric acid (H2SO4) absorbent for the phentate
method when interferences are present. An intensely blue compound, indophenol, is
formed by the reaction of ammonia, hypochlorite, and phenol catalyzed by sodium
nitroprusside.
An intensely blue compound, indophenol, is formed by the reaction of ammonia,
hypochlorite, and phenol catalyzed by sodium nitroprusside.
Complexing magnesium and calcium with citrate eliminates interference produced

by precipitation of these ions at high pH. There is no interference from other trivalent
forms of nitrogen. Interfering turbidity can be removed by distillation or filtration. If
hydrogen sulfide is present, it can be removed by acidifying samples to pH 3 with dilute
HCl and aerating vigorously until sulfide odor no longer can be detected.
Pipet Wash bottle
III. PROCEDURES:
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REAGENTS
1. Phenol solution:
Mix 11.1 mL liquified phenol (≥89%) with 95% v/v ethyl alcohol 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 substance.
2. Sodium nitroprusside, 0.5% w/v:
Dissolve 0.5 g sodium nitroprusside in 100 mL deionized water. Store this

solution in amber bottle for up to 1 month.
3. Alkaline citrate:
Dissolve 200 g trisodium citrate and 10 g sodium hydroxide in deionized

water. Dilute to 1000 mL.
Sodium hypochlorite, commercial solution, about 5%.:
This solution slowly decomposes once the seal on the bottle cap is broken.
Replace about every 2 months.
Oxidizing solution:
Mix 100 mL alkaline citrate solution with 25 mL sodium hypochlorite.

Prepare fresh daily.
8. Stock ammonium chloride solution:
Dissolve 3.819 g anhydrous NH4Cl (dried at 100°C) in water, and dilute to

1000 mL; 1.00 mL = 1.00 mg N = 1.22 mg NH 3.
9. Standard ammonium solution:
Use stock ammonium solution and water to prepare a calibration curve in a range
appropriate for the concentrations of the samples.
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3. Add 1 mL phenol solution, 1 mL sodium nitroprusside solution, and 2.5 mL
oxidizing solution with thorough mixing after each addition.
4. Cover samples with plastic wrap or paraffin wrapper film. Let color develop at room
temperature (22 to 27°C) in subdued light for at least 1 h. Color is stable for 24 h.
5. Measure absorbance at 640 nm.
1. Prepare individual calibration curves from a series of six standards within the
ammonia ranges by diluting stock ammonia solution into the sample concentration
range and treat these standards just like how the samples are treated.. Use a
distilled water blank with the combined reagent to make photometric readings for
the calibration curve.
Note: All solutions or samples containing the reagents must be poured into a waste
bottle after the experiment.
Sample / Absorbance mg/L NH3

Trial
Trial 1
Trial 2
Trial 3
Plot absorbance vs. ammonia concentration to give a straight line passing through
the origin and test at least one ammonia standard with each set of samples.
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VII. REFERENCES:
SOLORZANO, L. 1969. Determination of ammonia in natural waters by the
phenolhypochlorite method. Limnol. Oceanogr. 14:799.
PARSONS, T.R., Y. MAITA & C.M. LALLI. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press, Elmsford, N.Y.
Experiment No: 9
Title: DETERMINATION OF SULFATES CONTENT OF WATER/WASTEWATER BY

TURBIDIMETRIC METHOD
TLO#8: Execute the standard procedures in determining Sulfates content of water.
I. INTRODUCTION:
Sulfates is widely distributed in human nature and may be present in natural waters in
concentration ranging from few hundred to several thousand mg/L. Sulfates occur
naturally in numerous minerals, including barite (BaSO 4), epsomite (MgSO4•7H2O) and
gypsum (CaSO4•2H2O) (Greenwood &Earnshaw, 2984). These dissolved minerals
contribute to the mineral content of drinking waters.
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Sulfates and sulfuric acid products are used in the production of fertilizers, chemicals,
dyes, glass, paper, soaps, textiles, fungicides, insecticides, astringents and emetics. They
are also used in the mining, wood pulp, metal and plating industries, in sewage treatment
and in leather processing (Greenwood & Earnshaw, 1984). Aluminum sulfate (alum) is
used as a sedimentation agent in the treatment of drinking-water. Copper sulfate has
been used for the control of algae in raw and public water supplies (McGuire et al., 1984).
Sulfates are discharged into water from mines and smelters and from kraft pulp and
paper mills, textile mills and tanneries. Sodium, potassium and magnesium sulfates are
all highly soluble in water, whereas calcium and barium sulfates and many heavy metal
sulfates are less soluble. Atmospheric sulfur dioxide, formed by the combustion of fossil
fuels and in metallurgical roasting processes, may contribute to the sulfate content of
surface waters. Sulfur trioxide, produced by the photolytic or catalytic oxidation of sulfur
dioxide, combines with water vapor to form dilute sulfuric acid, which falls as “acid rain”
(Delisle& Schmidt, 1977).
The Environmental Protection Agency (EPA) standards for drinking water fall into two
categories -- Primary Standards and Secondary Standards. Primary Standards are
based on health considerations and are designed to protect people from three classes of
toxic pollutants -- pathogens, radioactive elements and toxic chemicals. Secondary
Standards are based on taste, odor, color, corrosivity, foaming and staining properties of
water. Sulfate is classified under the secondary maximum contaminant level (SMCL)
standards. The SMCL for sulfate in drinking water is 250 milligrams per liter (mg/L),
sometimes expressed as 250 parts per million (ppm).
Sulfate may have a laxative effect that can lead to dehydration and is of special
concern for infants. With time, people and young livestock will become acclimated to the
sulfate and the symptoms disappear. Sulfur-oxidizing bacteria pose no known human
health risk.
Turbidimetric Method
The turbidimetric method of measuring sulfate is based upon the fact that barium
sulfate tends to precipitate in a colloidal form and that this tendency is enhanced in
presence of a sodium chloride—hydrochloric acid solution containing glycerol and other
organic compounds. The absorbance of the barium sulfate solution is measured by a
nephelometer or turbidimeter and the sulfate ion concentration, determined by
comparison of the reading with a standard curve.
The turbidimetric method is also based on the fact that light is scattered by particulate
matter in aqueous solution. When barium and sulfate react in water, they make the
solution turbid, which means the concentration of the sulfate can be measured by using
a spectrophotometer. The equation for the reaction of barium and sulfate is shown below
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SO42-(aq) + Ba2+(aq) -----> BaSO4(s)
Pipet Wash bottle
III. PROCEDURES:
REAGENTS
1. Sodium hydroxide, NaOH, 1 N:
Weigh and dissolve sufficient amount of NaOH pellets in distilled water to

prepare a desired volume of 1 N NaOH solution. Standardize against potassium
hydrogen phthalate (KHP) solution.
2. Sulfuric acid solution, H2SO4, 0.02 N:

a desired volume of 0.02 N H2SO4
3. Standard Calibrating Solution 1:
Fill the 100 mL volumetric flask with small amount of distilled water, add
10.4 mL 0.02 N H2SO4 and dilute to the mark. Cover the flask and shake the
solution.
Standard Calibrating Solution 2:
Weigh and dissolve 0.1479 grams of anhydrous Na2SO4 in small amount of

distilled water in a beaker. Transfer the solution to 1000 mL volumetric flask and
dilute to the mark. Cover the flask and shake the solution. The solution contains
100 microgram/L.
Buffer Solution:
Weigh and dissolve 30 grams of magnesium chloride in 500 mL distilled water.

Add 5 grams of sodium acetate, 1 gram of potassium nitrate and 20 mL of 99%
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grade acetic acid. Stir and homogenize the solution. Transfer the solution to 1000
mL volumetric flask and dilute to the mark.

3. Add 20 mL buffer solution and one spoonful of barium chloride.
4. Stir the sample for 1 minute and let it stand for 5 minutes.
5. Measure the absorbance from the spectrophotometer set at 420 nm wavelength.
1. Prepare several concentrations of standard sulfate solution (5, 10, 15, 20, 30 and
40 mg/L) and follow the same procedure for sulfate determination in
water/wastewater sample.
Note: All solutions or samples containing combined reagent must be poured into a
waste bottle after the experiment.
Sample / Absorbance mg/L SO4-

Trial
Trial 1
Trial 2
Trial 3
Plot absorbance vs. phosphate concentration to give a straight line passing

through the origin and test at least one phosphate standard with each set of samples.
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VII. REFERENCES:
Experiment No: 10
Title: DETERMINATION OF CHEMICAL OXYGEN DEMAND OF

WATER/WASTEWATER BY REACTOR DIGESTION METHOD
TLO#11: Demonstrate the methods in determining the Chemical Oxygen Demand

(COD) of water.
I. INTRODUCTION:
The COD is defined as the number of oxygen equivalents consumed in the oxidation
of organic compounds by strong oxidizing agents, such as dichromates and
permanganates, and is indicative of the amount of organic pollutants present in the tested
sample.
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
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chemicals such as ammonia and nitrite. COD measurements are commonly made on
samples of waste waters or of natural waters contaminated by domestic or industrial
wastes. Chemical oxygen demand 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
sulfuric acid (H2SO4). 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), another standard test for assaying the oxygen-demanding strength of
waste waters. However, biochemical oxygen demand only measures the amount of
oxygen consumed by microbial oxidation and is most relevant to waters rich in organic
matter. It is important to understand that COD and BOD do not necessarily measure the
same types of oxygen consumption. For example, COD does not measure the oxygen-
consuming potential associated with certain dissolved organic compounds such as
acetate. However, acetate can be metabolized by microorganisms and would therefore
be detected in an assay of BOD. In contrast, the oxygen-consuming potential of cellulose
is not measured during a short-term BOD assay, but it is measured during a COD test.
COD is used as a general indicator of water quality and is an integral part of all water
quality management programs. Additionally, COD is often used to estimate BOD
(Biochemical Oxygen Demand) as a strong correlation exists between COD and BOD,
however COD is a much faster, more accurate test.
The most common COD method is the wet chemistry method. This involves a two
hour digestion at high heat under acidic conditions in which potassium dichromate acts
as the oxidant for any organic material present in a water sample. Silver sulfate is present
as the catalyst and mercuric sulfate acts to complex out any interfering chloride. Following
the digestion, the extent of oxidation is measured through indirect measurement of
oxygen demand via electrons consumed in the reduction of Cr6+ to Cr3+. This can be done
by titration or spectrophotometry.
COD Reactor Pipetol

Colorimeter Wash bottle
Pipet
III. PROCEDURES:
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REAGENTS
Pre-ordered Digestion Solution
1. Homogenize 500 mL of sample for 2 minutes in a blender. (Or Simply shake the
sample for 2 minutes)
2. Turn on the COD Reactor. Preheat to 150oC. Place the plastic shield in front of the
reactor.
3. Remove the cap of a COD Digestion Reagent Vial for the appropriate range.
Sample concentration range; mg/L COD Digestion Reagent Vial Type

0 – 150 Low Range
0 – 1500 High Range
0 – 15000 High Range Plus
4. Hold the vial at 45o angle. Pipet 2.00 mL (0.2 mL for the 0 – 15000 mg/L range) of
sample into the vial.
5. Replace the vial cap tightly. Rinse the outside of the COD vial with deionized water
and wipe the vial clean with a paper towel.
6. Hold the vial by the cap and mix over a sink. Invert gently several times to mix the
contents. Place the vial in the preheated COD Reactor.
7. Heat the vials for 2 hours.
8. Turn the reactor off. Wait for about 20 minutes for the vials to cool to 120 oC or less.
9. Invert each vial several times while still warm. Place the vial into a rock. Wait until
the vials have cooled to room temperature.
10. Measure COD using colorimeter. (Colorimetric Method)
11. Read the COD (in mg/L) of the sample in the colorimeter.
Sample / COD, mg/L

Trial
Trial 1
Trial 2
Trial 3
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VII. REFERENCES:
Experiment No: 11
Title: DETERMINATION OF BIOCHEMICAL OXYGEN DEMAND OF

WATER/WASTEWATER BY IODIDE-AZIDE METHOD
TLO#9: Demonstrate the procedures in determining the amount of Dissolved Oxygen

(DO) present in water.
TLO#10: Execute the standard procedures in determining the Biochemical Oxygen
Demand (5-day BOD) of water.
I. INTRODUCTION:
Biological/Biochemical Oxygen Demand is a widely used technique to express the

concentration of organic matter in waste water samples. It is a measure of the amount of
dissolved oxygen used by microorganisms in the water. If the amount of organic matter
in sewage is more, the more oxygen will be utilized by microorganisms to degrade
dumping sewage which containing high BOD value. Digestion of these organic
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compounds in neutral ecosystem such as lakes, rivers etc. can deplete available oxygen
and result in fish asphyxiation.
In the presence of free oxygen, aerobic bacteria use the organic matter found in
wastewater as “food”. The BOD test is an estimate of the “food” available in the sample.
The more “food” present in the waste, the more Dissolved Oxygen (DO) will be required.
The BOD test measures the strength of the wastewater by measuring the amount of
oxygen used by the bacteria as they stabilize the organic matter under controlled
conditions of time and temperature.
The BOD test is used to measure waste loads to treatment plants, determine plant
efficiency (in terms of BOD removal), and control plant processes. It is also used to
determine the effects of discharges on receiving waters. A major disadvantage of the
BOD test is the amount of time (5 days) required to obtain the results.
When a measurement is made of all oxygen consuming materials in a sample, the

result is termed “Total Biochemical Oxygen Demand” (TBOD), or often just simply
“Biochemical Oxygen Demand” (BOD). Because the test is performed over a five day
period, it is often referred to as a “Five Day BOD”, or a BOD5.
In many biological treatment plants, the facility effluent contains large numbers of
nitrifying organisms which are developed during the treatment process. These
organisms can exert an oxygen demand as they convert nitrogenous compounds
(ammonia and organic nitrogen) to more stable forms (nitrites and nitrates). At least
part of this oxygen demand is normally measured in a five day BOD. Sometimes it is
advantageous to measure just the oxygen demand exerted by organic (carbonaceous)
compounds, excluding the oxygen demand exerted by the nitrogenous compounds. To
accomplish this, the nitrifying organisms can be inhibited from using oxygen by the
addition of a nitrification inhibitor to the samples. The result is termed “Carbonaceous
Biochemical Oxygen Demand”, or CBOD.
BOD bottles Graduated cylinder

Incubator Buret
Erlenmeyer flask Buret clamp
Pipet Iron Stand
Pipetol Wash bottle
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III. PROCEDURES:
REAGENTS
1. Phosphate Buffer Solution:
Dissolve 8.5 g of KH2PO4, 21.75 g of K2HPO4, 33.4 g of Na2HPO4·7H2O,

and 1.7 g of NH4Cl in about 500 mL of distilled water and dilute to 1 L.
2. Magnesium sulfate 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 in distilled water and dilute to 1 L.
4. Ferric chloride solution:
Dissolve 0.25 g of FeCl3·6H2O in distilled water and dilute to 1 L.
5. Ammonium chloride solution:
Dissolve 1.15 g of NH4Cl in 500 mL of distilled water and dilute to 1 L.

Adjust the pH to 7.2 using NaOH solution. The solution contains 0.3 mg N/mL.
6. Starch solution:
Dissolve in 100 mL distilled water 2 g of the laboratory grade soluble starch

and 0.2 grams of salicylic acid. Heat the solution for faster dissolution of the
reagents and cool to room temperature.
7. Alkali-iodide-azide solution:
Dissolve 500 grams of NaOH pellets and 135 grams of NaI in distilled water.
Dissolve in 40 mL deionized water 10 grams of NaN3 and add this to the previous
solution prepared. Dilute to 1000 mL.
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8. Standard Sodium thiosulfate titrant:
Dissolve 6.205 g of Na2S2O3·5H2O, add and dissolve 0.4 g of NaOH pellets.

Transfer the solution into a 1 L volumetric flask and dilute to 1000 mL.
9. Dilution water:
Use deionized distilled water in making sample dilutions.
a. Preparation of Dilution Water
1. Aerate 5 L of deionized distilled water long enough to allow the water to become
saturated with dissolved oxygen (approximately 8 mg/L at room temperature). This
can be accomplished by aerating with clean compressed air.
2. Siphon into a separate container an amount slightly greater than will be needed
for the sample dilutions if less than the entire bottle will be used in a single day.
3. Add 1 mg/L each of phosphate buffer, magnesium sulfate solution, calcium
chloride solution and ferric chloride solution into the aerated water.
4. If nitrification inhibition is used, store seeded dilution water at 20°C long enough
for the dilution water depletion to meet the quality criteria (depletion of no more
than 0.2 mg/L DO). Storage is not recommended when nitrification inhibition is not
going to be used because nitrifying bacteria can develop in the dilution water
during storage.
5. If nitrification inhibition is to be used, add enough nitrification inhibitor to the dilution
water to produce a final concentration of 10 mg/L. As an alternative, 3.33 mg of
nitrification inhibitor can be added to each BOD bottle for inhibition.
b. Treatment of the Sample
Initial DO Concentration of the Sample
1. Prepare three incubation bottles containing 300 mL dilution water.

2. Remove 100 mL of dilution water from each of the incubation bottle and add 100
mL of water sample to each of the incubation bottle. Shake the bottle to mix the
content.
3. Add 1 mL MnSO4, 1 mL alkali-iodide-azide and 1 mL H2SO4 to each of the
incubation bottle.
4. Mix the resulting solution using pipet until the solution turned to yellow color.
5. Pipette 200 mL from each of the incubation bottle to Erlenmeyer flasks and titrate
with Na2S2O5 until a pale straw color appears. Note the volume of the titrant used.
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6. Add starch solution to obtain a dark blue solution.

7. Titrate the dark blue solutions with Na2S2O5 until the solutions become clear.
Record the volume of the titrant used.
Final DO Concentration of the Sample

2. Remove 100 mL of dilution water from each of the incubation bottle and add 100
mL of water sample to each of the incubation bottle. Shake the bottle to mix the
content.
3. Add 1 mL MnSO4 and 1 mL alkali-iodide-azide to each of the incubation bottle.
4. Mix the solution using a pipet until the solution turned to yellow color.
5. Cover the bottles tightly and place it inside the incubator at 20 oC for 5 days.
6. After 5 days, remove the incubation bottles from the incubator.
7. Add 1 mL H2SO4 to each of the incubator bottle and mix the solution.
Initial DO Concentration of the Dilution Water

2. Add 1 mL MnSO4, 1 mL alkali-iodide-azide and 1 mL H2SO4 to each of the
incubation bottle.
3. Mix the resulting solution using pipet until the solution turned to yellow color.
Final DO Concentration of the Dilution Water

2. Add 1 mL MnSO4 and 1 mL alkali-iodide-azide to each of the incubation bottle.
3. Mix the solution using a pipet until the solution turned to yellow color.
4. Cover the bottles tightly and place it inside the incubator at 20 oC for 5 days.
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5. After 5 days, remove the incubation bottles from the incubator.

6. Add 1 mL H2SO4 to each of the incubator bottle and mix the solution.
Sample / DOi, mg/L DOf, mg/L BOD5, mg/L

Trial
Trial 1
Trial 2
Trial 3
 Standardization of Na2S2O3:
mmol Na2S2O3 = mmol potassium bi-iodate solution
 Determination of BOD:
BOD, mg/L = DOI - DOF

P
where: DOI - DO concentration in sample before incubation

DOF – DO concentration in sample after incubation
P - decimal fraction of wastewater sample used;
(volume of wastewater) / (vol. of dilution water plus wastewater)
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VII. REFERENCES:
Experiment No: 12
Title: WATER/WASTEWATER MICROBIOLOGICAL TEST BY MULTIPLE TUBE

FERMENTATION TECHNIQUE (MTFT)
TLO#12: Measure the total coliform and fecal coliform present in water.
I. INTRODUCTION:
Water microbiology is concerned with the microorganisms that live in water, or can be
transported from one habitat to another by water. Water can support the growth of many
types of microorganisms. This can be advantageous. For example, the chemical activities
of certain strains of yeasts provide us with beer and bread. As well, the growth of some
bacteria in contaminated water can help digest the poisons from the water.
However, the presence of other disease causing microbes in water is unhealthy and
even life threatening. For example, bacteria that live in the intestinal tracts of humans and
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other warm blooded animals, such as Escherichia coli, Salmonella, Shigella, and Vibrio,
can contaminate water if feces enter the water. Contamination of drinking water with a
type of Escherichia coli known as O157:H7 can be fatal. The intestinal tract of warm-
blooded animals also contains viruses that can contaminate water and cause disease.
Bacteria are introduced into waters from many sources naturally or by man and his
activities. Feces from warm-blooded animals, including humans, may, at any time, contain
disease-producing microbes consisting of bacterial pathogens, viruses, or internal
parasites. Most bacteria present in surface waters are not harmful to health, but if
pathogenic organisms are ingested, disease or sickness may occur. Some of these
bacteria are the E. coli, total coliform and fecal coliform.
Escherichia coli (E coli) refers to one of the species of bacteria in the fecal coliform
group. It is found in large numbers in the gastrointestinal tract and feces of humans and
warm-blooded animals. Its presence is considered indicative of fresh fecal contamination,
and it is used as an indicator organism for the presence of less easily detected pathogenic
bacteria (similar to fecal coliform – typically used in assessment of drinking water).
Fecal coliform are bacteria found in the bodily waste of all warm-blooded humans and
animals, most species are not capable of survival outside the body for a long period of
time. Their presence in water indicates contamination by human sewage or animal
droppings (similar to E coli – typically used in assessment of wastewater).
Total coliform are a group of bacteria found in soil, on vegetation, and in large
numbers in the intestine of warm-blooded animals, including humans. Water is not natural
medium for coliform organisms and their presence in water is an indication of some type
of contamination. Most coliform bacteria are not disease-causing organisms, but they
serve as an indicator of the sanitary conditions of the water supply.
One of the more important laboratory tests to determine water quality is the
bacteriological test. This test indicates whether or not a given water is bacterially
contaminated, and the extent of such contamination. The test is critically in public supply
systems where bacteria may cause an outbreak of disease, however, in surface waters
the test is usually not quite as critical, though it may be equally as important to the user.
In addition to the laboratory test, other information concerning the probable source and
significance of the count must also be obtained in order for the analysis to be meaningful.
Multiple Tube Fermentation Technique (MTFT)
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The multiple-tube procedure is one of the best tested and most authenticated
microbial test procedures and is used as a basis for water quality standards. This
procedure involves a series of preliminary and confirmatory tests in which gas bubbles
are formed in small glass vials by the action of coliform bacteria in a lactose broth medium.
A statistical analysis, called the most-probable-number (MPN), is then made. The MPN
is not an actual count of organisms, but merely on ideas of the number of coliform bacteria
which, more probably than any other number, would give results shown by the laboratory
examinations.
Incubator Medicine dropper

Autoclave Bunsen burner
Culture tube with Durham tube Iron stand
Pipet Iron ring
Pipetol Wire gauze
Beaker Wash bottle
III. PROCEDURES:
REAGENTS
1. Lactose Broth:
For the lactose broth which is a base for the cultivation of salmonella and
coliform organisms, suspend 13.0 grams powder in a 500 mL of distilled water and
mix thoroughly. Warm gently until the solution was completed. Distribute 20 mL
each in culture tubes containing inverted Durham tube and sterilize by autoclaving
the solution at 121oC for 15 minutes. Afterwards, cool the broth quickly.
2. Brilliant Green Bile Broth:
Suspend 40.01 grams of the BGBB powder in 1000 mL of distilled water

and heat to dissolve the medium completely. Distribute 20 mL each in culture tubes
containing inverted Durham tube and sterilize by autoclaving the solution at 121oC
for 15 minutes. Afterwards, cool the broth quickly.
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3. EC Broth:
Suspend 37.0 grams of the powder in a 1500 mL and heat to dissolve the
medium completely. Distribute 20 mL each in culture tubes containing inverted
Durham tube and sterilize by autoclaving the solution at 121 oC for 15 minutes.
Afterwards, cool the broth quickly.
1. Pour 10 mL of the water sample in each of the culture tube containing 20 mL lactose
broth (presumptive broth). 5 culture tubes are used per sample.
2. Shake gently until the sample was uniformly distributed throughout the medium. Make
sure that there is no bubble inside the inverted Durham tube after shaking the solution.
3. Incubate the tubes for 24 hours at 35oC.
4. After 24 hours, examine the tubes for the presence of gas seen in the Durham tube
or effervescence which denote positivity of result. Repeat the incubation for another
24 hours when the initial outcome was vague.
5. After 48 hours, transfer 2 drops from each presumptive tube into EC medium broth
and BGBB using sterilized pipette.
6. For the confirmation of total coliforms, incubate the BGBB from each of the
presumptive positive tube for 24 hours at 35oC. After the incubation period, check the
tubes for the presence of gas production and/or effervescence indicating positivity of
result.
7. To confirm for the presence of thermotolerant coliforms, incubate the EC tubes from
each of the presumptive broth for 24 hours at 44oC and check for the positivity of
results basing from gas production after the incubation period.
Sample / Lactose BGBB EC Broth

Trial Broth Broth
Trial 1
Trial 2
Trial 3
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Trial 4
Trial 5
Compute using Thomas’ simple formula the Most Probable Number (MPN) of coliform
organisms present in the water sample using the MPN index.
VII. REFERENCES:
Philippine National Standards for Drinking Water – 1993
COURSE #: CHE 3131L

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LABORATORY MANUAL
Experiment No: 13
Title: DETERMINATION OF HEAVY METALS OF WASTEWATER SLUDGE BY

TOXICITY CHARACTERISTICS LEACHING PROCEDURE (TLCP)
TLO#1: Identify and account for the standard laboratory guidelines and procedures
I. INTRODUCTION:
The occurrence of heavy metals in the environment is of important concern due to

their toxicity and health effects on humans, including cancer. Because metals have been
extensively used for centuries in commerce, environmental contamination is widespread;
moreover, exposure to metals and metal compounds continues to be a significant public
health problem.
Because of the toxicity to humans and plants, heavy metals (HMs) and metalloid
contaminants released in the environment are of major concern worldwide. This toxicity
COURSE #: CHE 3131L

59
LABORATORY MANUAL
is lethal even in trace quantities as metals have a great tendency to bio accumulate.
Environmental exposure to HMs is a well-known risk factor for cancers. The presence of
these contaminants in our environment is increasingly due to industrial pollution of the
atmosphere, waterways, soils and sediments. Tons of these elements are released into
atmosphere by burning of the fossil fuels, smelting and other processing techniques,
which can be carried long distances and later deposited on vegetation and soil. Therefore,
there are many sources of them. All are daily ingested by humans either through the air
or through food, water and soil. Metals have been extensively used for centuries in
commerce; consequently, environmental contamination is widespread and exposure to
metals and metal compounds continues to be a significant public health problem. This
would be true for people consuming grain grown on cadmium-enriched soil, from either
phosphate fertilizer or sewage sludge, for people consuming fish from mercury-enriched
lakes, or for those consuming vegetables from a lead-smelting contaminated area.
Toxicity Characteristics Leaching Procedure (TLCP)
Toxicity characteristic leaching procedure (TCLP) is a well-known index for

assessment of the hazards of different metals according to US EPA SW-846 method
13119 to evaluate the toxicity of a substance. For example, Tang and colleagues, studied
pre-treatment of tannery sludge with phosphoric acid (PA) and monobasiccalcium
phosphate (MCP) as a potential heavy metal emission control option. This treatment was
shown to effectively stabilize Pb and Cd in the tannery sludge. Pb and Cd leachability, as
determined by TCLP leaching tests, decreased with increasing P amendment addition
rate by 32.6% and 44.7% for PA treatment and 40.1% and 39.5% for MCP treatment,
respectively
The Toxicity Characteristic Leaching Procedure (TCLP) is designed also to

determine the mobility of both organic and inorganic analytes present in liquid, solid, and
multiphasic wastes. The intent of this leachate procedure is to simulate the conditions
that may be present in a landfill where water may pass through the land- filled waste and
travel into the groundwater carrying the soluble materials with it. This procedure does not
apply to volatile organic analytes.
This Standard Operating procedure (SOP) is based on Environmental Protection

Agency (EPA) Methods SW846/1311and those requirements set forth in the latest
approved version of the National Environmental Laboratory Accreditation Committee
(NELAC) Quality Systems section.
COURSE #: CHE 3131L

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LABORATORY MANUAL
Oven Pipet
Erlenmeyer flask Pipetol
Evaporating dish Beaker
Hot plate Volumetric flask
III. PROCEDURES:
REAGENTS
1. Extraction fluid 1:
Add 5.7 mL glacial acetic acid to 500 mL distilled water. Also add 64.3 mL
1 N NaOH solution and dilute it to 1 L.
2. Extraction fluid 2:
Add 5.7 mL glacial acetic acid to distilled water and dilute to 1 L.
a. Moisture Determination
1. Weigh 10 grams of the sludge sample placed in an evaporating dish.

2. Dry the sample in an oven for 2 hours at 103 to 105 oC.
3. Compute the moisture content by taking the difference between the initial and final
weight.
b. Heavy Metals Determination
Determination/Procedures when volatiles were Not Involved (Method 1311

USEPA)
1. For liquid wastes (<0.5% dry Solids), extract the waste by filtering the liquid waste
through a 0.6 to 0.8 µm glass fiber filter, the extract is called the TCLP extract.
2. For wastes containing >0.5 % solids, separate the liquid from the solid phase and
store for later analyses (to be combined with the final extract)
3. Reduce the particle size to 1 mm (when necessary)
4. Clean the Extraction Bottle with nitric acid.
5. Weigh 100 grams of wet sludge and then filter it then analyze the filter.
COURSE #: CHE 3131L

61
LABORATORY MANUAL
6. Extract the solid phase with an amount of extraction fluid equal to 20 times the
weight of solid phase.
Extraction fluid:
Wt. of extraction fluid= 20 x percent solids x weight of waste filtered / 100
Determination of extraction fluid:
Weigh out a sub sample and reduce to size to 1mm in diameter or less then
transfer 5 grams of this sample to a 500 mL flask. Then add 96.5 ml of deionized
water and cover it with a watch glass. Stir vigorously for 5 minutes in a magnetic
stirrer.
Adjusting the pH
The pH will be greater than 5 so add 3.5 mL 1 N HCL, mix and heat to 50oC
for 10 minutes then the cool the solution to room temperature. Measure the pH
and if it’s greater than 5, use extraction fluid 1.
7. Extract the solid using appropriate fluid for 18 hours at a speed of 30 rpm.
After the extraction, separate the liquid extract using 0.6 to 0.8 µm glass fiber filters
and then mix the sample with the filtrate collected from the wet sludge and perform
metal analysis using ICP (Inductively Coupled Plasma).
c. Elutriation Test
1. Measure 100 grams of sample.

2. Dissolve the sample in 2 L distilled water. And agitate for 1 hour.
3. Separate the filtrate using 0.6 to 0.8 µm glass fiber filter.
4. Measure the metals by ICP.
d. Total Assay of Metals by Nitric Acid Digestion
1. Weigh out 2 grams of the sample.

2. Dilute the sample to 50 mL using distilled water.
3. Add 5 mL concentrated nitric acid.
4. Slowly boil the solution and evaporate about 10 mL on a hot plate.
5. Continue heating and adding nitric acid until digestion is complete. The end point
is a clear solution.
6. Wash the flask and filter when necessary and dilute with distilled water to 50 mL.
7. Measure the metal by ICP or any other appropriate methods.
Sample / Moisture Mass of Metal

Trial Content Sludge Detected
COURSE #: CHE 3131L

62
LABORATORY MANUAL
Trial 1
Trial 2
Trial 3
VII. REFERENCES:
COURSE #: CHE 3131L

63

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Saint Louis University

SCHOOL OF ENGINEERING AND ARCHITECTURE

Title: WATER CHARACTERIZATION AND DETERMINATION OF PHYSICAL and

At the end of this experiment, the student should be able to:

Water or wastewater sampling is generally performed by one of two methods, grab

COURSE #: CHE 3131L

Composite samples of effluent collected, stored, analyzed, tabulated and

Conductivity is a measure of water’s capability to pass electrical flow. This ability

The pH of a solution is measured as negative logarithm of hydrogen ion

COURSE #: CHE 3131L

II. EQUIPMENT/ MATERIALS NEEDED:

1 TDS Meter 1 Conductivity Meter

1. Proceed to your assigned sampling point/s.

COURSE #: CHE 3131L

B. Determination of Physical Water Quality Parameters:

a. Determination of the Temperature and pH of the Water Sample

1. Calibrate the pH meter using solutions of known pH.

b. Determination of the Conductivity of the Water Sample

c. Determination of the Turbidity of the Water Sample

d. Determination of the Total Dissolved Solids (TDS) of the Water Sample

COURSE #: CHE 3131L

5. Record the TDS displayed in the TDS meter.

IV. DATA AND RESULTS

Sample / pH Temperature Conductivity Turbidity Total

V. DISCUSSION (observation/ interpretation of results):

VI. CONCLUSION (option of the department if this part will be included):

Standard Methods for the Examination of Water and Wastewater

COURSE #: CHE 3131L

Title: DETERMINATION OF SOLIDS CONTENT OF WATER/WASTEWATER BY

At the end of this experiment, the student should be able to:

Solids refer to matter suspended or dissolved in water or wastewater. Solids may

COURSE #: CHE 3131L

TOTAL SUSPENDED SOLIDS AND TOTAL DISSOLVED SOLIDS

COURSE #: CHE 3131L

TOTAL FIXED SOLIDS, TOTAL VOLATILE SOLIDS AND SETTLEABLE SOLIDS

COURSE #: CHE 3131L

II. EQUIPMENT/ MATERIALS NEEDED:

Buchner filter Watch glass

b. Determination of the Total Solids of Water

COURSE #: CHE 3131L

1. Shake the water sample well

c. Determination of the Total Suspended Solids and Total Dissolved Solids of

1. Shake the water sample well.

1. Shake the water sample well.

COURSE #: CHE 3131L

COURSE #: CHE 3131L

IV. DATA AND RESULTS

Sample / TS TSS TDS TFS TVS

V. DISCUSSION (observation/ interpretation of results):

Where: A = weight of dried residue + crucible, mg at 104°C oven

Total Dissolved Solids

Where: A = weight of dried residue + crucible, mg at 104°C oven

COURSE #: CHE 3131L

Total Volatile Solids (Dissolve and Suspended Volatile Solids)

Total Fixed Solids (Fixed Dissolved and Fixed Suspended Solids)

Where: A = weight of dried residue + crucible, mg, before ignition

TSS = FSS + VSS

Where fixed suspended solids is computed by: