LABORATORY MANUAL FOR REAL SAMPLE ANALYSIS

(CHEM 4022)

PREPARED BY: KEDIR SEID MOHAMMED

OCTOBER 2023
Contents page
I. Introduction to The Chemical Laboratory ................................................................. III

II. Laboratory Safety Guide Lines .............................................................................. III

III. Sign and Symbols of Hazardousness of Chemicals ...............................................IV

1. Experiment-1 ............................................................................................................... 1

2. Experiment -2 .............................................................................................................. 3

3. Experiment -3 .............................................................................................................. 4

4. Experiment -4 .............................................................................................................. 7

5. Experiment -5 ............................................................................................................ 10

6. Experiment -6 ............................................................................................................ 12

7. Experiment -7 ............................................................................................................ 16

8. Experiment -8 ............................................................................................................ 19

9. Experiment -9 ............................................................................................................ 21

10. Experiment -10 ....................................................................................................... 23

11. Experiment -11 ....................................................................................................... 25

12. Experiment -12 ....................................................................................................... 28

IV. References .............................................................................................................. 31

I
Table of figures
Figure 1. Sign and symbols of hazardousness of chemicals ............................................IV
Figure 2. Apparatus and their name’s ........................................................................... VIII
Figure 3. Structure OF EDTA .......................................................................................... 16
Figure 4. Structure of Ascorbic acid (vitamin C) ............................................................. 19

II
 I. Introduction to the Chemical Laboratory
Protection of the health and safety of individuals in the laboratory and respect for
preservation of the environment are regarded by the Chemistry Department as moral
imperatives. A good safety program requires everyone to share the responsibility of staff,
and students.
Safety information will be provided in a number of ways. Each laboratory subject begins
with a mandatory safety lecture to provide general information and advice. In addition,
the instructions for each experiment and the accompanying teaching assistant, TA
presentations will contain safety information specific to each experiment. A list of basic
rules for safety in the laboratory, which you should be familiar with, is appended. It is
also imperative that you become familiar with your safety manual. Strict adherence to the
guidelines outlined in both of these references will promote a safe and successful lab
experience

II. Laboratory Safety Guide Lines

Accidents in a chemical laboratory usually result from improper judgment on the part of
the victim or one of his/her neighbours. Learn and observe the safety and laboratory rules
listed below.
 The safe way is the right way to do your job. Plan your work, follow instructions.
If you do not know how to do the experiment safely, ask your teaching assistant
(TA).
 Wear safety goggles. Because the eyes may be permanently damaged by
spilled chemicals and flying broken equipment,
Note: Eye washing with a contact lens in place will not clear a splashed chemical from
the eye. The contact must be removed for effective cleansing. It is advisable for those
wearing contacts to switch to glasses for the lab period.
 Eating, drinking, and smoking are strictly prohibited in the laboratory at all times
because of the possibility of chemicals getting into the mouth or lungs through
contamination. The chief hazard with smoking is fire. Store food in the
refrigerators in the laboratory is absolutely forbidden.

III
  Personal effects: wear proper clothing (including protective clothing when
handling corrosive, toxic, or flammable materials). Avoid loose sleeves, loose
cuffs, and bracelets. Be careful with long hair.
 Always add a reagent slowly--never "dump" in for two reasons:
A. Some reactions give off a lot of heat, and unless adding slowly, can become too
vigorous and out of control.
B. If you make a mistake and choose the wrong chemical, adding slowly decreases the
possibility of causing a serious accident.
 Clean up your workspace at the end of each laboratory period
A. Wash and wipe off your desktop.
B. Be sure gas and water are turned off.
C. Return all special equipment to the stockroom.
D. Put everything back into your locker drawer and lock

III. Sign and Symbols of Hazardousness of Chemicals

Most chemical used in lab have different level of hazardousness and here shown below is
a selection of the most commonly used GHS (Globally Harmonized System of
Classification and Labelling of Chemicals) Hazard Labels used in the BIOLABs to
denote potential health, physical and environmental hazards associated with certain
materials and equipment and draw attention to the need to take precautions with their use.
GHS is an internationally agreed upon system, created by the United Nations in 2000,
and designed to replace the various classification and labeling standards used in different
countries.
Globally Harmonized System (GHS) of Classification and Labelling of hazards

Figure 1. Sign and symbols of hazardousness of chemicals

IV
Oxidizer: Oxidizing substances can ignite flammable and combustible material
or worsen existing fire and thus make firefighting more difficult. Caution! Keep
away from flammable, combustible and spontaneously combustible materials
Poisonous Substances: Very hazardous to health when inhaled, swallowed or when they
come in contact with the skin. May even lead to death. Danger! Avoid contact with the
human body and immediately contact a physician in case of contact.
Apparatus and their name’s

Wire Gauze
Bunsen burner

Beakers

Tripod Stand

Burette
Test Tubes

V
 Funnels

Titration flasks

Evaporating Dish Graduated or Measuring cylinder

Pipette
Thermometer

Watch glasses
Iron stand

VI
 Test Tube Clamp
Wash Bottles

Beam Balance

Volumetric flask

Hot plate

Reagent bottle with dropper

Tong

Clamp

VII
Round bottom flask Condenser

Suction flask
Fractionating column

Three necked round bottom flask Buchner funnel

Figure 2. Apparatus and their name’s

VIII
LABORATORY REPORT FORMAT
This is the format that the students must follow when they write their lab reports.
The cover page must include the following components
Date of Submission, Institution Name, College, Department, Experiment Number, Group
number, Group Members List
NAME IDNO
1. ------------------------------------ --------------
2. ------------------------------------ --------------
3. ------------------------------------- -------------
4. ------------------------------------ ---------------
5. ------------------------------------ ---------------
Parts of the report
1. Title page
2. Abstract
3. Objective
4. Introduction
5. Apparatus and instruments
6. Procedure
7. Observation
8. Data and Calculations
9. Results and discussion
10. Conclusions
11. Reference

IX
 1. Experiment-1
Determination of water content
Objective
 To determine water content of soil samples
Theory
Soil is a material composed of five ingredients- minerals, soil organic matter, living
organisms, gas, and water. Soil minerals are divided into three size classes- clay, silt, and
sand.
Soil is a mixture of organic matter, minerals, gases, liquids, and organisms that together
support life. Earth's body of soil, called the pedosphere, has four important functions: as a
medium for plant growth as a means of water storage, supply and purification as a
modifier of Earth's atmosphere as a habitat for organisms. All of these functions, in their
turn, modify the soil and its properties. It is also commonly referred to as earth or dirt;
some scientific definitions distinguish dirt from soil by restricting the former term
specifically to displaced soil.
Most laboratory tests in soil mechanics require determination of water content. water
content is defined as
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑤𝑎𝑡𝑒𝑟 𝑝𝑟𝑒𝑠𝑒𝑛𝑡 𝑖𝑛 𝑎 𝑔𝑖𝑣𝑒𝑛 𝑠𝑜𝑖𝑙 𝑚𝑎𝑠𝑠
𝑤=
𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑠𝑜𝑖𝑙
Apparatus and chemicals
 Moisture can(s): moisture cans are available in different size
 Oven with temperature control: for drying the temperature of an oven generally
kept between 105oc-1100c. a higher temperature should be avoided to prevent
burning of organic matter in soil
 Balance: the balance should have readability of 0.01g for specimens having 200g
or less. If the specimen has mass over 200g the readability should be 0.1 g.
 soil
Procedure
 Determine mass (g) of empty moisture can plus its cap (w1), and also record the
number, place a sample of representative moist soil in a can and close the can
with its cap to avoid loss of moisture.

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  Determine the combined mass (g) of closed can and moist soil (w2). Remove the
cap from top of the can and place it at the bottom of the can. Put the can (step
above) in oven to dry the soil to a constant weight.
 Determine the combined mass (g) of dry soil sample plus the can and its cap (w3).
Calculations
Calculate the mass of moisture, W2-W3
Calculate the mass of dry soil, W3-W1
Calculate the water content
𝑊2−𝑊3
W (%) = × 100
𝑊3−𝑊1

Report the water content to the nearest 1% or 0.1% as appropriate based on the size of
specimen.

2
 2. Experiment -2
Moisture content determination of food samples
Objective
 To determine the percentage of water in a sample by drying the sample to a
constant weight
Theory
Moisture content refers to the number of water molecules that become incorporated into
a food product. Moisture can enter into a product in a number of ways, it could be related
to the production method of the product, the atmospheric moisture in the food production
area, the packaging method of the product, or it can be related to the method of food
storage.
Moisture content influences the taste, texture, weight, appearance, and shelf life of
foodstuffs. Even a slight deviation from a defined standard can adversely impact the
physical properties of a food material. For example, substances which are too dry could
affect the consistency of the end product. Conversely, excess moisture may cause food
material to agglomerate or become trapped in the piping systems during production.
Also, the rate of microbial growth increases with total water content, possibly resulting in
spoiled batches that need to be disposed of. However, water is also an inexpensive
ingredient adding to the weight of the final product. Hence, obtaining an optimal
analytical value for moisture is of great economic importance to a food manufacturer.
Apparatus and chemicals
 Drying equipment (oven, hot plate)
 Drying dish
 Analytical balance
 Desiccator containing desiccant and
 Sample foods
Procedure
Weight evaporating dish and moisture sample immediately and record as “wet weight of
sample (W1)“. Dry the weight sample to a constant weight at the temperature not
exceeding 239 ℉ (115 ℃) using the suitable drying equipment. Allow the sample to cool
and weigh the cooled sample again and record as the “dry weight of sample (W2)”.
Finally, the moisture content can be determined through the equation
𝑊1
Moisture content =(𝑊2) ∗ 100

3
 3. Experiment -3
Determination of Calcium by Permanganate Titration
Objective
 To determine calcium content of sample using redox titration method
Theory
In this experiment conducted, redox titration method is used in the analysis of an
unknown sample containing calcium. Redox titration is a laboratory technique used to
determine the concentration of a given analyte by causing a redox reaction between the
titrant and the analyte which in this case the standardized solution of potassium
permanganate, KMnO4 is titrated against the analyte sodium oxalate solution, Na2C2O4.
This reaction can be represented by the following ionic equation:
Reduction half equation: 𝑛𝑂4− + 8𝐻++ 5𝑒−→ 𝑀𝑛2+ + 4𝐻2𝑂
Oxidation half equation: 2𝑂42− → 2𝐶𝑂2 + 2𝑒 –
5𝐶2𝑂42−+ 2𝑀𝑛𝑂4− + 16𝐻+→ 2𝑀𝑛2+ + 10𝐶𝑂2+ 18𝐻2𝑂
According to the equation, MnO4 is reduced to Mn2+while C2O42– is oxidized to CO2. It
can also be seen that 2 moles of MnO4 - ion reacts with 5 moles of oxalate ion. Since the
reaction took place in an acidic medium, the oxidizing power of KMnO4 is increased. In
this case, the use of KMnO4 as the oxidizing agent in this experiment is quite useful as it
acts as its own indicator. The initial bright purple colour of KMnO4 is discharged due to
its reduction by oxalic acid. After oxalate ions are completely consumed, the end point is
indicated by the appearance of a light pink colour that persists from the addition of
excess unreacted KMnO4.
Apparatus and chemicals
 Food sample
 Deionized water
 0.02 M potassium permanganate (KMnO4) solution
 0.5 M sulfuric acid (H2SO4) solution
 Erlenmeyer flask
 Burette
 Pipette
 Funnel
 Analytical balance
 Burette clamp

4
  Bunsen burner
Procedure
1. Prepare the 0.02 M potassium permanganate (KMnO4) solution by dissolving an
appropriate amount of KMnO4 in deionized water. Use an analytical balance to
measure the required mass.
2. Set up the burette by rinsing it with the KMnO4 solution and then filling it up.
Note the initial volume reading of the burette.
3. Weigh an appropriate amount of the food sample using an analytical balance and
transfer it to an Erlenmeyer flask.
4. Add 50 mL of 0.5 M sulfuric acid (H2SO4) solution to the Erlenmeyer flask
using a pipette and funnel.
5. Heat the mixture gently on a Bunsen burner using a heat-resistant mat until it
begins to boil. Continue heating for 3-5 minutes to ensure complete digestion of
the organic matter.
6. Allow the mixture to cool to room temperature.
7. Rinse the burette with the KMnO4 solution again and refill it if necessary.
8. Titrate the cooled mixture with the KMnO4 solution, gradually adding it to the
flask while swirling continuously. The KMnO4 acts as the titrant in this reaction.
9. Observe the color change of the solution from purple to colorless to indicate the
endpoint of the reaction.
10. Record the final volume reading of the burette.
11. Repeat steps 7-10 for additional trials of the food sample, ensuring consistency
in the techniques used.
12. Calculate the moles of KMnO4 consumed based on the volume used during the
titration. Use the balanced chemical equation between KMnO4 and calcium to
determine the mole ratio between them. Calculate the moles of calcium present
in the food sample by multiplying the moles of KMnO4 consumed by the mole
ratio determined in step 13. Convert the moles of calcium to grams using the
molar mass of calcium (40.08 g/mol). Finally; calculate the percentage of
calcium in the food sample by dividing the mass of calcium by the initial mass of
the sample and multiplying by 100.
Data Analysis
For each trial, record the initial and final volume readings of the KMnO4 solution in the
burette. Calculate the average moles of KMnO4 consumed by summing the moles from

5
all trials and dividing by the number of trials. Calculate the average mass of calcium in
the food sample by summing the masses from all trials and dividing by the number of
trials. Calculate the percentage of calcium for each trial and then determine the average
percentage of calcium in the food sample.

6
 4. Experiment -4
Determination of iodine value of fats and oils
Objective
 to determine the iodine value of fats and oils
Theory
The iodine value is a crucial parameter used to assess the quality and chemical
composition of fats and oils. It provides important information about the degree of
unsaturation or the presence of double bonds within the fatty acid molecules in a given
sample. By determining the iodine value, we can gain insights into the nutritional value,
oxidative stability, and potential uses of fats and oils in various industrial applications,
such as food processing, biodiesel production, and cosmetic formulation.
Unsaturated fatty acids contain one or more double bonds in their carbon chain, which
result in a relatively lower melting point and a higher susceptibility to oxidation
compared to saturated fatty acids. The iodine value reflects the amount of iodine in grams
that can react with 100 grams of a fat or oil. When iodine reacts with the double bonds
present in the unsaturated fatty acids, the resulting compound is known as iodine
monochloride (ICl). By measuring the volume of iodine monochloride solution required
to react completely with the unsaturated bonds in a fat or oil sample, the iodine value can
be determined.
Understanding the iodine value is crucial for several reasons. Firstly, it helps in
evaluating the nutritional quality of fats and oils. For instance, oils with high iodine
values tend to contain a higher proportion of essential fatty acids, such as omega-3 and
omega-6, which are important for various physiological processes in the human body.
Secondly, the iodine value provides valuable information about the stability and shelf life
of fats and oils. Fats with higher iodine values are generally more susceptible to oxidative
rancidity, leading to off-flavors and reduced nutritional value over time. On the other
hand, oils with lower iodine values are more stable and resistant to oxidation, making
them suitable for prolonged storage or high-temperature cooking.

Lastly, the iodine value plays a significant role in the production of biodiesel fuels.
Biodiesel is typically synthesized by the transesterification of vegetable oils or animal
fats with alcohol, such as methanol or ethanol. The iodine value helps determine the
degree of unsaturation in the feedstock oil, which affects the quality and combustion

7
characteristics of the resulting biodiesel fuel. High iodine values indicate higher levels of
unsaturated fatty acids, leading to lower oxidative stability and increased susceptibility to
polymerization during storage.
Apparatus and chemicals
 Fats and oils (samples to be tested)
 Iodine monochloride (ICl) solution
 Glacial acetic acid
 Potassium iodide (KI) solution
 Sodium thiosulfate (Na2S2O3) solution
 Starch indicator solution
 Glass stoppered flask
 Volumetric flask
 Burette
 Pipette
 Burette clamp
 Magnetic stirrer with stir bar
 Funnel
 Beaker
 Graduated cylinder
 Analytical balance
Procedure
Preparation of Solutions:
Prepare a 0.1 N iodine monochloride (ICl) solution by dissolving an appropriate amount
of ICl in glacial acetic acid. The concentration of ICl will depend on the expected iodine
value of the fats and oils being tested. Prepare a 10% potassium iodide (KI) solution by
dissolving 10 grams of KI in deionized water, using a volumetric flask. Stir the solution
until all the KI is dissolved. Prepare a 0.1 N sodium thiosulfate (Na2S2O3) solution by
measuring the required amount of Na2S2O3 using a pipette and then diluting it with
deionized water in a volumetric flask. Stir the solution until the Na2S2O3 is completely
dissolved. Prepare a starch indicator solution by adding a few drops of starch solution to
a small beaker of boiling water. Stir the mixture until the starch is uniformly dispersed.
Titration Procedure:
1. Weigh approximately 0.2-0.3 grams of the fat or oil sample using an analytical
balance, and record the mass.

8
 2. Transfer the weighed sample into a glass stoppered flask.
3. Add 10 mL of glacial acetic acid to the flask, followed by 25 mL of the prepared
0.1 N ICl solution.
4. Place the flask on a magnetic stirrer and adjust the stirring speed to ensure
thorough mixing and prevent the sample from settling.
5. Titrate the solution with the 0.1 N Na2S2O3 solutions until the yellow color
disappears and the solution turns pale yellow.
6. Add a few drops of starch indicator solution to the flask to observe the formation
of a blue color, indicating the near end-point.
7. Continue the titration dropwise until the blue colour disappears, signifying the
end-point of the reaction.
8. Record the volume (in mL) of the Na2S2O3 solution used for the titration.
9. Repeat steps 1-8 for at least three trials using different fat or oil samples.
10. Calculate the average volume of Na2S2O3 used for each sample.
Data Analysis:
Calculate the iodine value of each fat or oil sample using the following formula: Iodine
Value = (Volume of Na2S2O3 used × Normality of Na2S2O3) / Mass of Fat or Oil Sample
Calculate the average iodine value for all trials.
Compare the iodine values obtained for different fats and oils to determine their relative
levels of unsaturation.

9
 5. Experiment -5
Determination of Salt Content of Dairy Products (Volhard Method)
Objective
 To determine the salt content, specifically chloride ions (Cl-), in various dairy
products using the Volhard method.
Theory
The salt content of dairy products plays a significant role in determining their taste,
quality, and nutritional value. Salt, primarily in the form of chloride ions (Cl-), not only
enhances the flavor profile but also affects the texture and shelf life of dairy products.
Therefore, it is crucial to measure and monitor the salt content to ensure the desired
sensory attributes and product stability. The Volhard method is a commonly used
analytical technique for the precise determination of chloride ions in dairy products. This
method involves the precipitation of chloride ions with a silver nitrate solution, followed
by titration of the unreacted silver ions with ammonium thiocyanate. By utilizing the
Volhard method, accurate measurements of chloride ion concentration can be obtained,
providing valuable information about the salt content of dairy products.
Apparatus and chemicals
 Dairy product samples (e.g., milk, cheese, yogurt)
 Distilled water
 Silver nitrate solution (0.1 M)
 Ammonium thiocyanate solution (0.1 M)
 Potassium chromate indicator solution
 Burette
 Erlenmeyer flask
 Glass stirring rod
 Pipette
 Bunsen burner or hot plate
 Analytical balance
 Filtration apparatus (filter paper, funnel)
Procedure
Preparation of Silver Nitrate Solution:
Prepare a 0.1 M solution of silver nitrate by dissolving the appropriate amount in distilled
water. Ensure that the solution is accurately standardized before use.

10
Sample Preparation:
Weigh an appropriate amount (around 10g) of the dairy product sample using an
analytical balance. Note down the weight.
Precipitation of Chloride Ions:
 Transfer the weighed sample into a clean Erlenmeyer flask.
 Add 50 mL of distilled water to the flask and stir the mixture using a glass stirring
rod until the sample is fully dissolved.
 Add a few drops of potassium chromate indicator solution to the flask.
Titration:
 Slowly add the silver nitrate solution from the burette into the Erlenmeyer flask
while continuously stirring the mixture.
 Titrate until a reddish-brown precipitate of silver chromate appears, indicating the
complete reaction of chloride ions.
 The appearance of the reddish-brown color is the endpoint of the titration. Record
the volume of silver nitrate solution used.
Titration of Unreacted Silver Ions:
 After reaching the endpoint, add 5 mL of ammonium thiocyanate solution to the
flask. The solution should turn a reddish colour.
 Continue titration by adding the ammonium thiocyanate solution drop by drop
until the reddish colour disappears, indicating the complete reaction of unreacted
silver ions.
 Record the volume of ammonium thiocyanate solution used.
 Calculation of Salt Content:
 Calculate the chloride ion concentration using the volume of silver nitrate and
ammonium thiocyanate solutions used in the titration.
 Determine the salt content in the dairy product sample based on the chloride ion
concentration and the weight of the sample.

11
 6. Experiment -6
Analysis of BOD, DO in waste water sample
Objective
 To analyse BOD, DO in waste water sample
Theory
The analysis of biochemical oxygen demand (BOD), chemical oxygen demand (COD),
and dissolved oxygen (DO) in wastewater samples is a critical component of
understanding water quality and ensuring environmental stewardship. BOD serves as a
measure of the biodegradable organic matter present in water, reflecting the potential for
oxygen depletion due to microbial activity. COD, on the other hand, denotes the overall
quantity of oxygen required for chemical oxidation of organic and inorganic compounds,
providing insights into the pollution load in the water.
Additionally, the measurement of DO offers valuable information about the level of
oxygen available for aquatic life, highlighting the potential impact of the water on
ecosystems and downstream users.
The assessment of these parameters is instrumental in identifying the potential
environmental impact of wastewater discharges, understanding the health of aquatic
systems, and facilitating informed decision-making for water resource management. As
such, the accurate and precise analysis of BOD, COD, and DO in wastewater samples is
pivotal for ensuring compliance with environmental regulations, safeguarding public
health, and preserving natural ecosystems.
Apparatus and cheemicals
 Wastewater samples
 BOD bottles
 Graduated cylinders
 BOD incubator
 COD digestion vials
 Spectrophotometer
 Dissolved oxygen meter
 potassium dichromate
 sulfuric acid
 ferrous ammonium sulfate
 Alkaline-iodide-azide solution

12
  Manganese sulphate
 Starch solution
 0.025N sodium thiosulphate
Procedure
Neutralization of Sample:
o Take 50 ml of the water sample and place it in a 100 ml beaker.
o Measure the pH of the sample using a calibrated pH meter.
o Adjust the pH to 7.00 ± 0.2 using 1N sulfuric acid if it is greater than 7.00 or 1N
sodium hydroxide if it is less than 7.00.
o Note the volume of sulfuric acid or sodium hydroxide used to neutralize the 50 ml
sample to pH 7.00 ± 0.0.
o Calculate the amount of sulfuric acid or sodium hydroxide needed to neutralize
the 1000 ml sample.
o Add the specified volume of sulfuric acid or sodium hydroxide to neutralize the
sample
Removal of Chlorine Content:
 Take a 50 ml water sample in a conical flask for analysis.
 Add 2.5 ml of 50% diluted acetic acid and 2.5 ml of a 10% w/v potassium iodide
solution.
 Add 1 ml of indicator for starch and titrate with 0.025N sodium sulfite solution.
 Note the volume and calculate the amount to add to the 1000 ml sample as done
in the Neutralization of Sample section.
 Add the determined volume of sodium sulphite solution to the sample to
neutralize the chlorine.
Preparation of Phosphate Buffer Solution:
 Dissolve 8.5 grams of potassium dihydrogen phosphate (KH2PO4), 21.75 grams
of dipotassium hydrogen phosphate (K2HPO4), 33.4 grams of disodium hydrogen
phosphate (Na2HPO4.7H2O), and 1.7 grams of ammonium chloride (NH4Cl) in
500 ml of distilled water.
 Dilute the solution to a volume of 1 liter.
Preparation of Alkali-Iodide-Azide Reagent:
 Dissolve 500 grams of sodium hydroxide (NaOH) and 135 grams of sodium
iodide (NaI) in distilled water to make 1000 ml solution.
 Dissolve ten grams of sodium azide in the solution.

13
Preparation of Dilution Water:
 Take five liters of double-distilled water and air-condition it using clean
compressed air for a minimum of 12 hours.
 Allow the water to stabilize for at least six hours at 20°C.
 Add 5 ml each of 27.5% w/v calcium carbonate solution, 22.5% w/v solution of
magnesium sulphate, and ferric chloride 0.15% w/v solution.
 Add five milliliters of the phosphate buffer solution and mix thoroughly.
 Let the mixture stand for two hours.
Determination of the Biological Oxygen Demand of Water:
 Add 10 ml of the water sample to two of the 300 ml BOD bottles and fill the
remaining space with dilution water.
 Fill the remaining two BOD bottles with dilution water for the blank sample.
 Seal the bottles immediately without any air bubbles inside.
 Incubate one sample and one blank bottle at 20°C for five days.
 Analyze the remaining one sample and one blank vial of dissolved oxygen (DO)
immediately.
 Analyze the bottles incubated for 5 days for DO.
Test for Dissolved Oxygen (DO)
 Add 2 ml of a 36.4% manganous sulphate (MnSO4.H2O) solution to the water
sample by placing the pipette tip into the sample to allow oxygen to enter the
solution through droplets of solution.
 Use the same technique to add 2 ml of the alkali-iodide-azide reagent to the
sample.
 Allow the solutions to react with the dissolved oxygen in the sample.
 Once the precipitates have settled at the bottom, add 2 ml of strong sulfuric acid
near the surface of the sample.
 Thoroughly mix to dissolve the precipitates.
 Transfer 203 ml of the BOD sample to an Erlenmeyer flask.
 Titrate promptly with 0.025N Sodium Thiosulfate solution using a Starch
indicator until the blue hue fades, and note the burette reading.
 Perform a similar procedure to determine the burette reading for the blank
sample.
 Calculation of BOD
 Use the BOD formula to calculate the BOD value:

14
BOD = (DO1 - DO2) * Dilution Factor / Volume of Sample
 The final step in the BOD test procedure requires reporting the BOD value in
mg/L. It indicates the amount of oxygen consumed by microorganisms during the
test period. This value represents the organic pollution level of the water sample.

15
 7. Experiment -7
Determination of water hardness
Objective
 To determine the total hardness, permanent hardness, and temporary hardness of a
given water sample by complexometric titration (EDTA method)
Theory
The simple definition of water hardness is the amount of dissolved calcium and
magnesium in the water. Hard water is high in dissolved minerals, largely calcium and
magnesium.
Water that has not been purified is what is known as “hard water.” Hard water can
contain substances like Ca2+, Mg2+, and Fe2+. These “hard ions” are not always unhealthy
necessarily, but there are several good reasons that we remove them. First, they can
combine with other compounds to form soap scum. Second, it can lead to the build-up of
scale in pipes which may require costly repairs. Finally, the scale and soap scum will lead
to more expensive energy bills and more repairs needed in the long run
The amount of hard ions in water can be determined by the process of titration. In this
case, the titration is done by adding the chemical EDTA, ethylene-diamine tetra acetic
acid, to the water (which has a few drops of the indicator Erichrome Black T) until the
indicator changes from red to blue. The point at which the colour changes is called the
endpoint.

Figure 3. Structure OF EDTA

Temporary hardness
Temporary hardness is a type of water hardness caused by the presence of dissolved
bicarbonate minerals (calcium bicarbonate and magnesium bicarbonate). When
dissolved, these type of minerals yield calcium and magnesium cations (Ca2+, Mg2+)
and carbonate and bicarbonate anions (CO2−3 and HCO−3). The presence of the metal

16
cations makes the water hard. However, unlike the permanent hardness caused by sulfate
and chloride compounds, this "temporary" hardness can be reduced either by boiling the
water, or by the addition of lime (calcium hydroxide) through the process of lime
softening. Boiling promotes the formation of carbonate from the bicarbonate and
precipitates calcium carbonate out of solution, leaving water that is softer upon cooling.
Permanent hardness
Permanent hardness (mineral content) is generally difficult to remove by boiling. If this
occurs, it is usually caused by the presence of calcium sulphate/calcium chloride and/or
magnesium sulphate/magnesium chloride in the water, which do not precipitate out as the
temperature increases. Ions causing permanent hardness of water can be removed using a
water softener, or ion exchange column.
permanent hardness = permanent calcium hardness + permanent magnesium
hardness.
It is often desirable to soften hard water. Most detergents contain ingredients that
counteract the effects of hard water on the surfactants. For this reason, water softening is
often unnecessary. Where softening is practiced, it is often recommended to soften only
the water sent to domestic hot water systems so as to prevent or delay inefficiencies and
damage due to scale formation in water heaters. A common method for water softening
involves the use of ion exchange resins, which replace ions like Ca2+ by twice the
number of mono cations such as sodium or potassium ions.
Washing soda (sodium carbonate, Na2CO3) is easily obtained and has long been used as a
water softener for domestic laundry, in conjunction with the usual soap or detergent.
Apparatus and chemicals
 Burette
 Conical flask
 Stand
 Clamp
 Beaker
 0.1,0.05 N EDTA
 Sample water
 Ammonia-ammonium chloride buffer
 EBT indicator
Procedure to estimation of total hardness

17
Take 0.1N EDTA in a burette, Pipette out 20 ml of sample water into a conical flask, add
5 ml of freshly prepared ammonia buffer (PH=10) and add 2-3 drops of EBT indicator
then titrate the wine red solution with EDTA until sky blue colour appears (end point),
finally, repeat the titration to get concordant value and let the volume of EDTA be V1
ml.
Procedure to estimation of permanent hardness
Take 0.05 N EDTA in a burette, boil the water sample gently for 10 minutes, Cool and
filter and Pipette out 20 ml of sample water into a conical flask then add 5 ml of freshly
prepared ammonia buffer and add 2-3 drops of EBT indicator then titrate the wine red
solution with EDTA until steel blue colour appears. Finally, repeat the titration to get
concordant value and let the volume of EDTA be V2 ml.
NOTE; Ammonia/Ammonium chloride buffer stock solution, pH 10 is prepared by
dissolving 64 g of ammonium chloride in 600 ml of concentrated ammonia (14.8,28%
NH3) and add 400 ml distilled water slowly and carefully with stirring.
Calculations
Total hardness
Ht=V.EDTA *C. EDTA/V. Water
Where Ht is the total hardness, in mmol /L ;
V1 is the volume of EDTA solution consumed during titration in mL
C is the molar concentration of EDTA in mol/L
V2 is the volume of water used for determination of hardness, in mL
Use Same calculation for permanent hardness

18
 8. Experiment -8
Analysis of Vitamin-C using iodine
Objective
 To determine the amount of vitamin C in samples, such as lemon
Theory
Vitamin C (ascorbic acid) is an antioxidant that is essential for human nutrition. Vitamin
C deficiency can lead to a disease called scurvy, which is characterized by abnormalities
in the bones and teeth. Many fruits and vegetables contain vitamin C, but cooking
destroys the vitamin, so raw citrus fruits and their juices are the main source of ascorbic
acid for most people.
One way to determine the amount of vitamin C in food is to use a redox titration. The
redox reaction is better than an acid-base titration since there are additional acids in a
juice, but few of them interfere with the oxidation of ascorbic acid by iodine.

Figure 4. Structure of Ascorbic acid (vitamin C)

One of the most important micronutrients of lemon juice is vitamin C. Vitamin C is a
sensitive antioxidant and heat, light and oxygen are included important factors to reduce
the amount of this vitamin. This study designed to evaluate vitamin C in industrial lemon
juice quantitatively. The present cross- sectional study was conducted on 50 bottles of
different types of Shiraz industrial lemon juice. Vitamin C levels were measured through
iodine titration method.
Apparatus and chemicals
 Beaker
 Balance
 Stirrer
 Conical flask
 Burette
 Stand

19
  Clamp
 Starch for preparing starch indicator
 Distilled water
 Iodine for preparing starch indicator
 Lemon
 Ascorbic acid
Procedure
A) Preparing solutions
Add 0.5 g soluble starch to 50 near boiling distilled water, mix well and allow to cool
before use. Weigh 1.6 g of iodine and add it into 250 ml beaker and add 250 ml of
distilled water and swirl for a few minutes until iodine is dissolved
B) Titration
Weigh the lemon and extract the lemon juice, pipette 10 ml aliquot of the sample solution
in to 250 ml conical flask and add about 30 ml of distilled water and 2 ml of starch
indicator solution. Titrate the sample with 0.05 mol/l iodine solution. the end point of the
titration is identified as the first permanent trace of a dark blue-black colour due to the
starch - iodine complex
Calculations
Weight of lemon =
Volume of lemon juice=
Volume of lemon juice + water (V2 of sample before dilution) =
Concentration of I2 (C1) =0.05M
Volume of I2 that give end point (V1) =
C2 is concentration of ascorbic acid (AA)
C1V1=C2V2
C2=V1C1/V2-----------this is before dilution
But the concentration of ascorbic acid without water (C2) is C1V1=C2V2
Where C1 = conc. Of ascorbic acid with water as calculated above
V1 =volume of lemon juice only and water
V2 =volume of lemon juice only
% of AA =mass of AA/mass of lemon × 100

20
 9. Experiment -9
Determination of acetic acid in vinegar
Objective
 To determine the molarity and percent by mass of acetic acid in vinegar
Theory
Titrations are among the most commonly used and accurate methods of determining the
amount of analyte in an unknown solution. In this procedure you will be performing two
titrations, one to determine the concentration of the sodium hydroxide solution prepared
in Part I and another to determine the concentration of acetic acid in a sample of vinegar.
Each of these is an example of an acid-base titration. The standard solution may be
prepared in two ways – the direct or indirect method. In the direct method, a precisely
weighed quantity of the pure solute (primary standard) is dissolved and diluted to a
known volume in a volumetric flask. The concentration of the standard solution is then
calculated from the known mass of the solute and the known volume of the solution. If
the solute used to prepare the standard solution is pure and the solution is stable (does not
decompose), then the compound is referred to as a primary standard. However, often it is
not possible to obtain the solute in sufficiently pure form to be suitable as a primary
standard. For example, NaOH(s) reacts with gases (H2O and CO2) in the air which means
that NaOH (s) is not pure enough to be used as a primary standard. In this case the
standard solution is prepared by an indirect method. A solution is prepared at
approximately the desired concentration and it is then standardized against another
primary standard to determine its exact concentration.
Apparatus and chemicals
 Burette
 Volumetric flask
 Stand and Clamp
 Erlenmeyer flask
 Wash bottle
 Funnel
 NaOH
 Distilled water
 Vinegar
 Phenolphthalein indicator

21
Procedure
Rinse the inside of burette with distilled water and with small amount of NaOH(aq), Fill
the burette with NaOH (aq) up to top then transfer 5 ml of vinegar in to a clean 250 ml
Erlenmeyer flask and add about 20 ml of distilled water and 5 drops of phenolphthalein
to this Erlenmeyer flask. Titrate by slowly adding NaOH from the burette to the vinegar
in the Erlenmeyer flask. swirl Erlenmeyer flask as you add the base in order to efficiently
mix the chemicals. some pinkness may appear briefly in the flask as the base is added but
it will disappear quickly as the flask is swirled.
As the equivalence point is approached, the pink color will become more pervasive and
will take longer to disappear. when it occurs, start to add the NaOH drop by drop
eventually the addition of just one drop of NaOH will turn the solution in the Erlenmeyer
flask a pale pink colour that does not disappear while stirring. this indicates that the
equivalence point has been reached. measure the volume of NaOH precisely and finally,
refill your burette with NaOH and then repeat this procedure for second sample of
vinegar and then the third sample of vinegar
Calculation
Determine the volume of NaOH at equivalence point
Write the balanced equation of neutralization reaction and calculate the number of moles
of vinegar neutralize.
Calculate the number of grams of acetic acid in a vinegar
Calculate the percent of acetic acid in vinegar
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑
% of acetic acid in vinegar =𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑣𝑖𝑛𝑒𝑔𝑎𝑟 𝑠𝑜𝑙𝑢𝑡𝑖𝑜𝑛 × 100

22
 10. Experiment -10
Determination of chloride in water sample
Objective
 To determine the amount of chloride by using argentometric titration
Theory
The chloride ion is highly mobile and concentrations in water are not affected by
chemical reactions. Hence chloride does not biodegrade, readily precipitate, volatilize, or
bio accumulate. The chloride ion does not adsorb readily onto mineral surfaces and
therefore concentrations remain high in surface water and sediment pore water, and low
in sediment.
Mohr method (Argentometric method) was a very simple and highly selective method for
the determination of chloride ion (Cl-) using silver nitrate as the titrant.
The determination of chloride involves the use of silver nitrate in the presence of
potassium chromate as an indicator.
Apparatus and chemicals
 Conical flask
 Beaker
 Burette
 Erlenmeyer flask
 Stand and clamp
 Standard silver nitrate solution
 Indicator potassium chromate solution
 Tap water
Procedure
A) Titration with blank solution
Transfer 30 ml of tape water in a conical flask and add 3-4 drops of indicator potassium
chromate solution. slowly add standard 0.1 M silver nitrate solution from burette and
shake the solution well. at the end point light yellow colour starts changing to red colour.
the titration is repeated until a concordant volume V1 is obtained.
B) Titration with sample water
Transfer 30 ml of the given water sample in a conical flask and add 3-4 drops of indicator
potassium chromate solution. slowly add standard silver nitrate solution from the burette
and shake the solution well. At the end point, light yellow colour starts changing to red

23
colour and red colour persists. the titration is repeated until a concordant volume V2 is
obtained
Result
The amount of chloride ion in a given sample water ……………. ppm
𝑉𝐴𝑔𝑁𝑂3×𝑁×35.45×100
mg/L= 𝑚𝐿 𝑠𝑎𝑚𝑝𝑙𝑒

V2= mL titration for sample

V1= mL titration for blank
N= normality of AgNO3

24
 11. Experiment -11
Determination of pH in water samples
Objective
 To determine pH of given water sample
Theory
The term pH refers to the measure of hydrogen ion concentration in a solution and
defined as the negative log of H+ ions concentration in water and wastewater
pH=-log[H+]
Where [H+] is the concentration (or activity) of hydrogen ion (or photon) in moles per
litter (M). Water dissociates to form hydrogen ion (H+) and hydroxyl ion (OH-)
according to the following equation:
[H2O] = [H+] + [OH-]
At equilibrium, we can write,
[𝐻 + ]+[𝑂𝐻 − ]
KW= [𝐻2 𝑂]

But, since concentration of water is extremely large (approximately 55.5 mol/L) and is
diminished very little by the slight degree of ionization, may be considered as a constant
and its activity is taken as 1.0. Thus the above equation may be written as:
KW = [H+] + [OH-]
Where, Kw = Equilibrium Constant, For pure water at 25 °C, KW = 10-7×10-7= 10-14. This
is known as the ion product of water or ionization constant for water. In other words,
water (de-ionized or distilled water) at 25°C dissociates to yield 10-7mol/L of hydrogen
ion (H+) and 10-7mol/L of hydroxyl ion (OH-). Hence, according to Equation (1) pH of
deionized water is equal to 7. The values of pH,0 to a little less than 7 are termed as
acidic and the values of pH a little above 7 to 14 are termed as basic. When the
concentration of H+ and OH- ions are equal then it is termed as neutral pH.
Determination of pH is one of the important objectives in biological treatment of the
wastewater. In anaerobic treatment, if the pH goes below 5 due to excess accumulation of
acids, the process is severely affected. Shifting of pH beyond 5 to 10 upsets the aerobic
treatment of the wastewater. In these circumstances, the pH is generally adjusted by
addition of suitable acid or alkali to optimize the treatment of the wastewater. pH value
or range is of immense importance for any chemical reaction. A chemical shall be highly
effective at a particular pH. Chemical coagulation, disinfection, water softening and
corrosion control are governed by pH adjustment. Lower value of pH below 4 will

25
produce sour taste and higher value above 8.5 a bitter taste.
Higher values of pH hasten the scale formation in water heating apparatus and also
reduce the germicidal potential of chlorine. High pH induces the formation of tri-
halothanes, which are causing cancer in human beings. According to Bangladesh
Environment Conservation Rules (1997), drinking water standard for pH ranges from 6.5
to 8.5. It is done by using PH electrode The pH electrode used in the pH measurement is a
combined glass electrode. It consists of sensing half-cell and reference half-cell, together
form an electrode system. The sensing half-cell is a thin pH sensitive semi permeable
membrane, separating two solutions, viz., the outer solution, the sample to be analyzed
and the internal solution enclosed inside the glass membrane and has a known pH value.
An electrical potential is developed inside and another electrical potential is developed
outside, the difference in the potential is measured and is given as the pH of the sample.
Apparatus and chemicals
 pH meter
 Beaker
 Buffers Solutions of known pH value
 Sample water
Precautions
 The following precautions should be observed while performing this experiment:
Temperature affects the measurement of pH at two points. The first is caused by
the change in electrode output at different temperatures. This interference can be
controlled by the instruments having temperature compensation or by calibrating
the electrode instrument system at the temperature of the samples. The second is
the change of pH inherent in the sample at different temperatures. This type of
error is sample dependent and cannot be controlled; hence both the pH and
temperature at the time of analysis should be noted.
 In general, the glass electrode is not subject to solution interferences like color,
high salinity, colloidal matter, oxidants, turbidity or reductants.
 Oil and grease, if present in the electrode layer, should be removed by gentle
wiping or detergent washing, followed by rinsing with distilled water, because it
could impair the electrode response.
 Before using, allow the electrode to stand in dilute hydrochloric acid solution for
at least 2 hours.
 Electrodes used in the pH meter are highly fragile, hence handle it carefully.

26
Procedure
Three major steps are involved in the experiment. They are
1. Preparation of Reagents
2. Calibrating the Instrument
3. Testing of Sample
Generally, with simple procedure;
Perform calibration of the pH meter using standard pH solutions. The calibration
procedure would depend on the pH range of interest. In a clean dry 100 mL beaker take
the water sample and place it in a magnetic stirrer, insert the Teflon coated stirring bar
and stir well, now place the electrode in the beaker containing the water sample and
check for the reading in the pH meter. Wait until you get a stable reading. Take the
electrode from the water sample, wash it with distilled water and then wipe gently
Questions
Define pH in terms of hydrogen-ion (H+) concentration and hydroxyl-ion (OH-)
concentration. An increase in pH of one unit represents how much decrease in hydrogen
ion concentration?

27
 12. Experiment -12
Determination of Total Solids, Dissolved Solids and Suspended Solids in Water
samples
Objective
 To determine the total, dissolved and suspended solids in given water sample
Theory
Total Solids (TS) are the total of all solids in a water sample. They include the total
suspended solids and total dissolved solids. Total Suspended Solids (TSS) is the amount
of filterable solids in a water sample. Samples are filtered through a glass fiber filter. The
filters are dried and weighed to determine the amount of total suspended solids in mg/L
of sample.
Total Dissolved Solids (TDS) are those solids that pass through a filter with a pore size
of 2.0 micron (1/1000000th of a meter, also known as a Micro meter) or smaller. They
are said to be non-filterable. After filtration the filtrate (liquid) is dried and the remaining
residue is weighed and calculated as mg/l of Total Dissolved Solids.
Total solids measurements can be useful as an indicator of the effects of runoff from
construction, agricultural practices, logging activities, sewage treatment plant discharges,
and other sources. Total solids also affect water clarity. Higher solids decrease the
passage of light through water, thereby slowing more rapidly and hold more heat; this, in
turn, might adversely affect photosynthesis by aquatic plants. Water will heat up affect
aquatic life that has adapted to a lower temperature regime. As with turbidity,
concentrations often increase sharply during rainfall, especially in developed watersheds.
They can also rise sharply during dry weather if earth-disturbing activities are occurring
in or near the stream without erosion control practices in place. Regular monitoring of
total solids can help detect trends that might indicate increasing erosion in developing
watersheds.
Total solids are related closely to stream flow and velocity and should be correlated with
these factors. Any change in total solids over time should be measured at the same site at
the same flow. Water with total solids generally is of inferior palatability and may induce
an unfavourable physiological reaction. It may be aesthetically unsatisfactory for
purposes such as bathing. Total solids will be higher in highly mineralized waters, which
result in unsuitability for many industrial applications. It indicates effectiveness of
sedimentation process and it affects effectiveness of disinfection process in killing

28
microorganisms. It is used to assess the suitability of potential supply of
water for various uses. In the case of water softening, amount of total solids determines
the type of softening procedure. Corrosion control is frequently accomplished by the
production of stabilized waters through pH adjustment. The pH stabilization depends to
some extent upon the total solids present as well as alkalinity and temperature.
Suspended organic solids which are degraded anaerobically may release obnoxious
odours. Biologically active suspended solids may include disease causing organisms as
well as organisms such as toxic producing strains of algae. The suspended solids
parameter is used to measure the quality of wastewater influent and effluent. Suspended
solids determination is extremely valuable in the analysis of polluted waters.
The measurement of solids is by means of the gravimetric procedure. The various forms
of solids are determined by weighing after the appropriate handling procedures. The total
solids concentration of a sample can be found directly by weighing the sample before and
after drying at 103°C. However, the remaining forms, TDS and TSS require filtration of
the sample. For liquid samples, all these solids levels are reported in mg/L. A rapid
assessment of the dissolved solids content of water can be obtained by specific
conductance measurements. Such measurement indicates the capacity of a sample to
carry an electric current which in turn is related to the concentration of ionized
substances in the water. Most dissolved inorganic substances in water are in ionized form
and so contribute to the specific conductance. Although the nature of the various ions,
their relative concentrations, and the ionic strength of the water affect this measurement,
such measurement can give practical estimate of the dissolved mineral content of water.
The TDS content can be approximated by multiplying the specific conductance in micro-
Siemens per centi-meter (µS/cm) by an empirical factor varying from 0.55 to 0.90
depending on the chemical composition of the TDS.
Apparatus and chemicals
 Balance
 Beaker
 Filter paper
 Funnel
 Dropper
 Oven
 sample water

29
procedure
Measurement of Total Solids (TS)
Take a clear dry glass beaker (which was kept at 103℃ in an oven for 1 hour) of 150ml.
capacity and put appropriate identification mark on it. Weight the beaker and note the
weight. Then Pour 100ml. of the thoroughly mixed sample, measured by the measuring
cylinder, in the beaker and Place the beaker in an oven maintained at 103℃ for 24hours.
After 24 hours, cool the beaker and weight. Find out the weight of solids in the beaker by
subtracting the weight of the clean beaker determined in initially and finally, calculate
total solids (TS) as explained below:
Measurement of Total Dissolved Solids (TDS)
Same as above (step of total solids). Take a 100 mL of sample and filter it through a
double layered filter paper and collect the filtrate in a beaker. Then repeat the same
procedure as in steps of the total solids determination and determine the dissolved solids
contents as follows:
Calculation
Total solids, TS (mg/L) = mg of solids in the beaker x 1000 / (volume of sample)
Total Dissolved Solids, TDS (mg/L) =mg of solids in the beaker x1000 /(volume of
sample)
Total Suspended Solids, TSS (mg/L) = TS (mg/L) – TDS (mg/L)
Questions
1. Discuss possible sources of solids in ground water and surface water.
“Groundwater usually has higher dissolved solids and surface water usually has
higher suspended solids”- Explain.
2. Why water is evaporated at 103°C rather than 100 °C in assessment of solid of
water?

30
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