Analytical Chemistry 1

Sources of Information in Analytical Chemistry

You will learn several methods in analytical chemistry as you take up this course. These methods involve
steps that are crucial. It is important that the principle for these steps are fully understood. The principles
for these procedures can be found in standard analytical chemistry textbooks. The UST Library contains
numerous textbooks in analytical chemistry. These are located at the Science and Engineering Section (2nd
Floor).

Sometimes, textbooks may not be enough. There are times when procedures may need some
modifications depending on the type of sample. In this case, two of the most useful sources of information
are the Official Methods of Analysis of the AOAC and the Annual Book of ASTM Standards. These are
compilations of analytical methods and test procedures for various materials and chemicals. The
procedures in these books have passed through a rigorous process of validation and are generally accepted
to be the methods of choice. A brief description of the principles of the methods is also included in the
procedures. Both these books are available at the Reference Section of the Science and Engineering
Section of the UST Library.

Analytical chemistry is an actively evolving field of chemistry. Research on new analytical methods is
continuing. Thousands of research results are coming out every year. These researches attempt to solve
problems encountered in analytical chemistry. The results of these researches are published in scientific
journals. Our library has a good collection of these scientific journals. Some of these journals that may be
useful to you are:

Analytical Chemistry
Biosensors and Bioelectronics
Journal of Chemical Education
Journal of Biological Chemistry
Nature
Science

Aside from these journals, our library has access to journals that are published on line (i.e., the internet)
and downloadable as pdf files. These journals have been “peer reviewed”.

There are many journals available in the internet. Some of these require membership in certain
professional organizations or a fee. Some could be accessed for free. A list of these journals is given
below:

Analytical Chemistry – A publication of the American Chemical Society (http://pubs.acs.org/journals/ancham/)

Limnology and Oceanography – A journal on all aspects of oceanography including the analysis of some important
chemical parameters of water (http://aslo.org/lo/)

Journal of Biological Chemistry – A few old articles in this journal are the basis of some methods in analytical
chemistry (http://www.jbc.org/)
Analytical Chemistry 2

Volumetric Glasswares

Volumetric glasswares are specialized glasswares designed to contain (TC) or to deliver (TD) a FIX
AND EXACT volume of a liquid. The volume that these glasswares can contain or deliver is usually
written on the surface. A line marks the point where the level of the liquid should be.

Volumetric glasswares must be thoroughly cleaned before use. They should not be heated or be used for
hot liquids. If beads of water form inside these glasswares, they should be cleaned until the water inside
will form a continuous film when drained.

The Buret
The buret is a long, cylindrical glassware used to deliver (TD) exact
volumes of liquids. The buret has graduations (i.e. measurements)
etched on the side. The graduations usually start with 0.00 mL near
the top. A stopcock or pinchcock at the lower end controls the flow
of the liquid inside the buret. If the stopcock is made of glass, the
buret must not be used for highly alkaline liquids.

The lower meniscus of the liquid is used to “read” the volume of the
liquid if the liquid is clear.

The buret is cleaned with soap and water. Before using, the buret is
rinsed with the solution to be placed inside. This practice minimizes
the error incurred when the solution inside the buret gets diluted due
to the presence of water or some other reactive substances left on the
sides.

Before filling up the buret, make sure that the stopcock is closed.

The buret need not be filled up to the 0.00 mL mark. Make sure that
the tip below the stopcock is filled up with the solution. The volume
of the liquid that has been delivered by the buret is the difference
between the initial and the final volume.
http://core.ecu.edu/chem/chemlab/equipment/images/burette.jpg

The student must be careful when writing the data for the volume. The buret that will be used for Chem
300 is a 50 mL buret. It is capable of delivering 0.1mL with certainty. Since, all numerical data must have
one and only one uncertain digit, the data for the volume must be written to reflect one uncertain digit.
This means that the data for buret readings must have two decimal places.
Analytical Chemistry 3

The Volumetric Pipette

The volumetric pipette is used to deliver (TD) a fixed volume of liquid. It is characterized by a ‘bulge” in
the middle. Specific details on how to use a pipette can be obtained from these websites:

www.umd.umich.edu/casl/natsci/slc/ slconline/PIPET/PPT%20pipette%20slides-RLD4.PPT

www.sfu.ca/chemistry/students/ courses/chem126/Techniques/pipeting.htm

The illustration below shows the general steps in using a volumetric pipette:

The first step involves drawing in the solution

into the pipette. An aspirator bulb is used to
draw in the liquid by suction. Immerse the tip of
the pipette into the liquid. Squeeze the bulb first
before positioning it on the tip of the pipette.
Slowly release the aspirator bulb so that the liquid
is “aspirated” into the pipet. Allow the liquid to
run past, above the mark.

When the liquid has ran past the mark, remove the
aspirator bulb and stop the flow of the liquid by
placing the INDEX FINGER (NOT THE THUMB)
to close the opening. Lift the pipet above the container
of the liquid and wipe off any liquid outside the pipet.

With the pipette in a vertical position, the

liquid is allowed to drain until it reaches the
mark. This is done by slightly lifting the index
finger using the finger joint as a pivot
Analytical Chemistry 4

The contents of the pipette are then transferred

with the pipette at a vertical position. The tip of
the pipette is in contact with the surface of the
container. To do this, the container is slightly
tilted.

The Volumetric Flask

Volumetric flasks are used to prepare dilute solutions. A concentrated solution is introduced into a
volumetric flask usually by the use of a volumetric pipette. If a solid has to be dissolved, the solid is
weighed in a container (usually a beaker) then the solid is quantitatively transferred to the volumetric
flask by repeated rinsing. The solvent (usually water) is added until the total volume has reached the mark
or, in technical terms, diluted to volume. If the level of the solution exceeds that of the mark, the entire
process is repeated.

If a 10.00 mL portion of solution is introduced inside a 100.00 mL volumetric flask and the solution was
"diluted to volume", the resulting concentration of the new solution is exactly 1/10th of the original.

http://www.columbia.edu/itc/chemistry/chem-cf1500/labpix/images/volumetricflask.jpg
 Analytical Chemistry 5

The Analytical Balance and the Top Loading Balance

The basic function of balances is, simply, to determine the weight of a material.

The basic parts of balances are shown in the figure below:

Door
Weighing pan

Shield

Level indicator

Levelling screws

Power Switch
Read out
Tare Switch

The enclosure around the weighing pan is the main physical feature that differentiates an analytical
balance from a top loading balance. Modern analytical balances are easy to use because of the
incorporation of a microcomputer in its mechanism. Despite this, there are precautions that must be
followed so that the balance will give accurate and reproducible results.

It is a common misconception that the accuracy of a balance gets better if there are more digits on the
read out. THE ACCURACY OF THE DIGITAL ANALYTICAL BALANCE DOES NOT DEPEND ON
THE NUMBER OF DECIMAL PLACES IT COULD READ.

Accuracy is inherent in a balance (manufacturer, internal mechanism, model, design). If all things are
equal, then the accuracy and the reproducibility of a measurement will depend on how well a set of
standard operating procedures were followed. The following procedures are generally accepted as
standard procedures in operating balances:

1.) Make sure the weighing pan is clean and the doors of the analytical balance
are closed.
Analytical Chemistry 6

2.) Check the level indicator. The bubble on the level indicator should be within
the inscribed circle.

3.) The readout should show "0.00" if the weighing pan is empty.

4.) Generally, the object to be weighed must be in a container.

5.) Keep the door of the balance closed (if applicable).

6.) For high accuracy work, do not touch the surface of the object to be
weighed with your bare hands.

The balances are located inside the balance room. The temperature inside the balance room is kept
constant to insure that each weighing is done at a relatively constant temperature. If there are too many
people in the balance room, the environment may not be able to maintain a constant temperature. Because
of this, only a limited number of people are allowed inside the balance room.

To insure that users of the balances will find it convenient to weigh, there are some rules of “etiquette”
that must be followed:

l.) Do not unplug the balance.

2.) Clean the balance surface and vicinity.
3.) Close the doors of the balance after use.
4.) Make sure that the read out on the balance returns to 0.0000 after you have used the
balance.

The balances are for everybody’s use. Users of the balance expect that the balances are always ready for
use. It would be impolite if after using the balance, the next user finds the balance in disorder. Please,
always make sure that the next user will find the balance in the proper condition.

Logbook Keeping in Analytical Chemistry

In general, the logbook is a record of the data from and the conditions of an experiment. The reason for
keeping a record of the experiment is to insure that the same experiment could be repeated by a
reasonably competent person at a different place and time.

In our analytical chemistry course, most of the data are numerical. The conditions of an analysis are
always given in the procedure. This means that the logbook for analytical chemistry would mostly contain
numerical data or the results of the analysis.
Analytical Chemistry 7

Numerical data must be written such that any mathematical operation on these data could be made
simpler. For example if a data needs to be deducted from a previous data, then it would be easier if the
two data are written in a vertical column rather than in a horizontal row. An example is shown below.

Source: R.A. Day and A.L., Underwood, (1991) Quantitative Analysis 6 th ed., Prentice Hall International Editions.

Location in the
Title Manunal

Summary

The most important rule in logbook keeping is to WRITE ALL YOUR DATA DIRECTLY ON THE
LOGBOOK. Never write your data on a sheet of paper or on another notebook. Some students do not
write directly on the logbook because they want to keep their logbooks neat. That is not the way to keep
your logbook neat. The way to keep your logbook neat is to practice writing directly on your logbook.

Another rule in logbook keeping is NEVER ERASE ANY DATA. Incorrect data entries are not erased.
Instead, simply draw a single horizontal line over the incorrect entry.
Analytical Chemistry 8

Weighing and Data Analysis Exercise

Suppose a car is to be designed for Filipinos, what should be the height of the interior of this car? What weight
should the car be able to carry?

In order to answer these questions, a car designer must know the height and weight of a Filipino. But a quick look at
your classmates will tell you that not everybody has the same weight and height. To solve this problem, the designer
must decide on the most probable height and weight of most Filipinos. But what are the most probable height and
weight of the Filipino? How many Filipinos should be measured to get the most probable height and weight of the
Filipino?

A similar problem exists in analytical chemistry. Imagine a chemist who wants to know how much chlorine is
present in 10,000 liters of water stored in a water tank. At most, the chemist can only get 0.1 liter of water from the
water tank. From that 0.1 liter, the chemist will get only 0.010 liter (ten milliliters) for analysis. After the analysis is
over, the chemist will make a decision for 10,000,000 milliliters of water (or 10,000 liters) based on the single
analysis of 10 milliliters.

This exercise will teach you how a chemist can make a decision for the entire population based on the analysis of a
sample or a small percentage of the population.

In this exercise, you will be asked to weigh one peso coins. The data will then be mathematically manipulated. At
the end of the exercise you will report the most probable weight of the one peso coin.

Procedure

1.) Obtain 5 clean one peso coins.

2.) Place them on a clean sheet of paper.
3.) Place a container on the analytical balance. The container could be a sheet of clean paper or a
watchglass. The idea behind the container is that the coin should not be placed directly on the
balance pan.
4.) Press “TARE”. When you press the “TARE”, the readout should show 0.0000.
5.) Place 1 (one) one peso coin on (or in) the container. Do not handle the coin with your bare hands.
Record the mass.
6.) After the mass of the coin has been recorded, press the “TARE” button.
7.) Repeat steps #5 and #6 until all the coins have been weighed.

With the data you have obtained, compute the mean, standard deviation and the confidence interval (at the
95% confidence level).
Analytical Chemistry 9

An Introduction to the Determination of Fats and Oils by the Soxhlet Method

Fats and oils are generally soluble in solvents that are non – polar. If these non – polar solvents are poured
on samples containing fats and oils, the solvent will dissolve the fats and oils while the rest of the sample
will stay undissolved. If the solvent (containing the fats and oils) is separated from the sample (by
filtration), and the solvent is evaporated, the fats and oils remain behind. A residue of fats and oils will be
left behind. This residue can then be weighed and the amount of fats and oils determined.

The Soxhlet method determines the amount of fats and oils through the use of a Soxhlet Extraction Set
up (Figure 1).

Figure 1. Soxhlet Extraction Set Up. Figure Figure 2. Detail of the Extraction Chamber.
taken from http://www.epa.gov/esd/chemistry/org- The arrow shows the side arm. Figure taken
anal/images/faq/soxhlet.png from http://www.uicoglass.com/envirom/ui4210.jpg

One feature of the Soxhlet apparatus is that the sample and the solvent are placed in separate containers.

The solvent is placed in the boiling flask. On top of the boiling flask is an extraction chamber. Figure 2
is a close up of the extraction chamber. The sample is placed inside the extraction chamber.

When the boiling flask is heated, the solvent turns to vapor or evaporates. The vapor rises through the side
arm (indicated by the arrow in Figure 2) and goes into the condenser. Inside the condenser, the vapor
cools down and is converted back to liquid.

The liquid solvent drips into the extraction chamber. The result is that the extraction chamber slowly fills
up with the solvent. When the level of the solvent inside the extraction chamber fills up to the level of the
siphon arm, the solvent gets siphoned back into the boiling flask. In the process, the fats and oils of the
sample in the extraction chamber is transferred into the boiling flask. Inside the boiling flask, the solvent
evaporates again (the fats and oils do not evaporate) and the process repeats itself.

The advantage of the Soxhlet method is that “fresh” solvent is used to repeatedly extract the fats and oils
from the sample. Because this is done repeatedly, the fats and oils are removed almost completely.
Analytical Chemistry 10

Another advantage of this method is that it can easily be automated where one person can handle ten or
more analysis with little effort. The disadvantage of this method is that the solvent commonly used is not
so environment friendly. Furthermore, the solvent may extract a variety of other soluble materials.

An Exercise in Regression Analysis

There are some “things” that increase when something else increase. For example, if you carry ten kilos of rice from the
first floor to the third floor, your muscle will ache by a small amount. But if you carry fifty kilos of rice, then the amount of
“ache” you will feel is greater. In other words, there is a relationship between the weight of rice you carry and the amount
of ache you feel.

The relationship between two values (whether linear, curvilinear, etc.) can be determined by regression analysis.

In this exercise, you will perform a regression analysis on the mass of water and its volume.

NOTE: Before starting the experiment, it is recommended that a sufficient amount of tissue be placed on the
weighing pan of the analytical balance.

Procedure
1.) Obtain a 100.0 mL volumetric flask, 5 mL graduated pipette, a beaker with distilled water, thermometer,
and a tissue.
2.) Check the temperature of the distilled water.
3.) Place the volumetric flask on the analytical balance. TARE the weight of the flask.
4.) Close the side doors of the analytical balance. Open the top door of the analytical balance.
5.) Transfer 1.0 mL of distilled water into the volumetric flask using the graduated pipette. Close the top door
of the balance. Record the mass.
6.) TARE the balance.
7.) Repeat step #5 and #6 but transfer 3.0 mL, 5.0 mL, 7.0 mL and 9.0 mL
8.) Perform regression analysis on the data.

Temperature of water: ______________________

Volume (mL) Mass (grams)

1.0

3.0

5.0

7.0

9.0
Analytical Chemistry 11

Guide to Volumetric Analysis

Volumetric analysis involves the reaction of a substance, called a titrant, with the analyte. The volume
of titrant is measured with a glassware known as a buret. The amount of the titrant required to react with
the analyte is then used to determine the amount of the analyte present. This implies that the reaction
stoichiometry of the titrant and the analyte must be known. In addition, the exact concentration of the
titrant must be known. In gravimetric analysis, we don’t need to know the reaction stoichiometry of the
precipitating agent and the analyte nor do we need to know the exact concentration of the precipitating
agent.

Theory

If the titrant (T) reacts with the analyte (A) on a 1:1 stoichiometry, then when the reaction is complete:

(1) Moles T = Moles A

We know the concentration of the titrant, MT, and the volume required to react with the analyte, VT.
Since the definition of M (molarity) is:

(2) M = moles / liter

If we multiply the concentration of the titrant with the volume of the titrant required to react with the
analyte (in liters), we get:

(3) MT x VT = Moles T

If we substitute (3) in (1):

(4) MT x VT = Moles A

The number of moles of the analyte can be expressed as:

(5) Moles A = grams analyte / molecular weight analyte

If (5) is substituted in (4):

(6) MT x VT = grams analyte / molecular weight analyte

Rearranging (6) so that grams analyte is isolated on one side:

(7) Grams analyte = MT x VT x molecular weight analyte

In general, if the reaction stoichiometry is given by xT + yA  P, then we have:

(8) Grams analyte = ( y / x ) (MT ) (VT) (Molecular weight analyte)

Analytical Chemistry 12

Modified Winkler Method for Dissolved Oxygen Determination

(The procedure is adapted from: J.H. Kennedy (1984), Analytical Chemistry Practice, Harcourt Brace Jovanovich Publishers, p.90)

A body of water can support aquatic life if there is enough oxygen dissolved in the water. The amount of dissolved oxygen
(d.o.) in a body of water is an indication of the amount of biological activity. During the daylight hours, the amount of
dissolved oxygen generally increases due to the photosynthetic activity of plants. The amount of d.o. is measured by a
special equipment known as a dissolved oxygen meter. However, a more accurate but more cumbersome method of d.o.
determination is presented here. The method is known as the Winkler Method.

The Winkler Method involves the addition of Mn2+ to a measured volume of water sample. Under alkaline conditions, the
Mn2+ is converted to Mn3+ by the dissolved oxygen present in the solution:

O2(g)) + 4Mn(OH)2(s) + 2H2O  4Mn(OH)3(s)

A solution of KI is then added. The Mn3+ present oxidizes the I-1 to I2 under acidic condition:

2Mn(OH)3(s) + 2I-1 + 6H+  2Mn2+ + I2 + 6H2O

The I2 produced is titrated by standardized Na2S2O3:

I2 + 2S2O3-2  2I-1 + S4O6-2

From the volume and concentration of the Na2S2O3, the amount of d.o. can be computed based on the balanced equation
shown above.

The Winkler Method uses specialized collection bottles known as Winkler bottles or dissolved oxygen bottles. In this
laboratory analysis, the Winkler Method will be modified. Instead of using Winkler bottles, syringes (50 mL) will be used
to collect the sample.

A. Preparation of 0.1 M Na2S2O3 titrant (approximate)

Boil 250 mL of distilled water for at least 5 minutes. Cool the water and add approximately 6 - 7 grams of
Na2S2O3•5H2O. Dissolve the solid by stirring. Add a pinch of Na2CO3. Store the solution in an amber
reagent bottle.

B. Preparation of the Primary Standard

Dry approximately 1 - 2 grams of KIO3 (iodide free) at 160°C for 1 hour. This is best done by placing the
KIO3 in a glass – weighing bottle and heating in an oven (without any cover on the weighing bottle).
After drying, quantitatively transfer 0.6 - 0.7 grams ( 0.lmg) to a 250.0mL volumetric flask. The
technique for this operation is by "subtractive weighing". Dilute to volume.

C. Standardization of the Na2S2O3 titrant (Determination of the EXACT concentration)

(Note: In this method, the addition of the indicator is delayed until the reaction is almost complete)

Weigh approximately 2 grams KI in three 250 mL beakers. Add 10.00 mL aliquots of the KIO3 standard
solution to each beaker. Add approximately 10mL 1M HCl to each beaker. The solution should turn
violet. Swirl gently.

Titrate the resulting solution until it turns light yellow. Add approximately 5 mL starch. The titration
mixture should turn blue. Continue titrating until the blue color disappears. Compute the concentration of
the Na2S2O3 solution. This is the concentration of the STANDARDIZED Na2S2O3.
Analytical Chemistry 13

D. Preparation of the Titrant for D.O. Determination

Transfer 5.00 mL of the STANDARDIZED Na2S2O3 to a 250.0 mL volumetric flask. Dilute to volume.
Use this as the titrant for dissolved oxygen determination. Use immediately. Dispose after one day.

E. Dissolved Oxygen Determination

(Sampling and sample preparation should be done ON SITE)

Carefully and slowly draw the water sample into the 50 mL syringe (A). Be sure that no air bubble is
trapped inside the syringe. Continue drawing in the sample until the 50 mL mark (B). After the sample
has reached the 50 mL mark, expel the sample slowly until the 30 mL mark is reached (C).

30 mL

Water level

A B C

Rinse with distilled water and wipe the outside of the syringe dry (D). Dip the syringe into the manganese
reagent (E). Carefully draw in the manganese reagent until the 35 mL mark. Make sure there are no air
bubbles drawn in. Slowly mix the reagent in the syringe by carefully tipping the syringe repeatedly. Rinse
and wipe dry the outside of the syringe (F).

D E F

Draw in the basic iodide reagent up to the 40 mL mark in a similar manner as the manganese reagent.
Rinse and wipe dry the outside of the syringe.
Analytical Chemistry 14

Determination of the pKa of a Weak Acid

When a solution of a weak acid is titrated COMPLETELY with a strong base, the salt that is formed is the
conjugate base of the weak acid. For example, in the tiration of CH3COOH with NaOH (or by -OH)

CH3COOH + -OH  CH3COO- + HOH

when the reaction is 100% complete, all of the weak acid (CH3COOH) is consumed and converted to its conjugate base
(CH3COO-).

If the reaction is only 50% complete, then we have a situation wherein only half of the weak acid has been titrated while
the other half remains untitrated. In other words, the concentration of the weak acid is the same as the concentration of its
conjugate base. At this point, the Henderson - Hasselbach equation reduces to pH = pKa (see appendix for the derivation of
the Henderson – Hasselbach equation).

In this analysis, the pKa of a weak monoprotic acid will be determined by plotting the pH change for every increment of
NaOH added. The pH change will be monitored using a pH meter.

A. Preparation of the NaOH Titrant (approximately 0.1 M)

Boil approximately 250 mL distilled water in order to remove the dissolved carbon dioxide in the water.
Add 1 gram of NaOH to the freshly boiled water. Cool and store in a reagent bottle with a plastic cap.

B. Preparation of the pH Meter

Handle the pH electrode carefully. Remove the protective cap at the tip of the pH electrode. Rinse the tip
with a flowing stream of distilled water. Soak the tip in a pH 7.0 buffer. Allow the reading of the pH
meter to stabilize for approximately 1 minute.

Check the pH reading. If the pH reading does not correspond to the pH of the buffer, read the instructions
for calibration. BE SURE TO RINSE THE ELECTRODE TIP WITH DISTILLED WATER BEFORE
AND AFTER SOAKING THE ELECTRODE IN A SOLUTION.

Your instructor might tell you to perform a two – point calibration. In this case, two different buffers are
used. Ask the instructor for the pH of the other buffer.

C. Standardization of the NaOH Titrant (OPTIONAL)

Weigh three replicates of 0.1 - 0.2 grams ( 0.1 mg) of potassium acid phthalate or KHP (MW 204.22) in
three separate beakers. Add approximately 50 mL distilled water to each of the beaker and stir until all the
KHP is dissolved. Place the pH electrode in the KHP electrode. Take note of the initial pH (i.e., pH at
volume zero). Add the titrant increments of 0.5 mL. Take note of the corresponding pH for every addition
of the titrant. Compute the molarity of the NaOH solution up to the fourth decimal place. KHP and
NaOH react on a 1:1 ratio.

D. Preparation of the Sample

If the sample is a solid, dissolve 0.1 grams of the solid in approximately 25 mL distilled water. If the
sample is a liquid, ask the instructor for the volume of the sample to be used.
Analytical Chemistry 15

E. Determination of the pKa

Place the pH electrode in the sample solution (see the figure). Swirl the beaker slowly. Wait for the pH to
stabilize. Note the pH. Keep the electrode in the solution. Add NaOH in small increments. After each
increment, allow the pH to stabilize. Note the pH. Theoretically, the increments of NaOH added must be
small for higher accuracy. However, that is time consuming. As a compromise, add the NaOH in
increments of 0.5 mL.
Keep on adding the NaOH in 0.5 mL increments until there is a sudden rise in pH. At this point, add in
increments of 0.1 mL until the pH shows a gradual rise in pH. Tabulate your results in a table similar to
the one shown below:

Total volume pH
NaOH added

F. Treatment of Results

Plot the total volume of NaOH added (x-axis) versus the pH (y-axis). Determine the volume of NaOH at
the inflection point on the curve. Divide the volume by two. Determine the pH coordinate of the
quotient. The pH is equal to the pKa.

For a more advanced treatment of result, obtain the first derivative of the curve.

Source: R.A. Day and A.L. (1991), Underwood, Quantitative Analysis 6 th ed., Prentice Hall International Editions.
Analytical Chemistry 16

Spectrophotometric Determination of Phosphorous

Phosphorous is present in bodies of water in the form inorganic phosphate (example: PO 4-3) or organic phosphate
(example: in the form of a nucleic acid). Phosphorous in natural waters originate from the leaching of phosphates from
rocks and mineralization of dead aquatic plants. Phosphorous is essential to the growth of aquatic plants and animals. It is a
vital inorganic nutrient that could limit what plants or animals can live in a given body of water. However, excessive
amounts of phosphorous have been implicated in a process known as euthrophication. Much of the excess phosphorous in
aquatic environments originates from man-made activities or the so-called anthropogenic sources. Since the exact location
of anthropogenic sources cannot be pinpointed, they are also known as non-point sources.

The objective of this analysis is to determine the orthophosphate concentration of a natural body of water (river, lake, sea).
In this analysis, it would be more significant if the location of one of the sampling sites is away from a populated area and
then compare the result from a sampling site near a populated area.

NOTE:
Rinse all glasswares with a solution of H2SO4 (approximately 5M) prior to use to remove any trace
phosphates from the detergent used to wash the glasswares.

A. Antimony potassium tartrate, K(SbO)C4H4O (catalyst)

Dissolve 1.3715 g K(SbO)C4H4O in 500 mL distilled water. Store in a refrigerator.

B. Ammoniun molybdate, (NH4)6Mo7O24•4H2O

Dissolve 20 g (NH4)6Mo7O24•4H2O in 500 mL distilled water. Place in a plastic bottle. Store in a

refrigerator.

C. Ascorbic acid (reducing agent)

Dissolve 1.76 g ascorbic acid in 100 mL distilled water. Store in the refrigerator. Dispose after one week.

D. Sulfuric acid, 5M

Place 250 mL distilled water in a one liter beaker. Carefully add 70 mL concentrated sulfuric acid with
constant stirring. Allow the solution to cool. Then add distilled water until the total volume of the solution
reaches 500 mL.

E. Coloring reagent (Mixed reagent)

Prepare only when needed

Place 50 mL of 5M sulfuric acid in a 250 mL beaker. Slowly add 5 mL of the K(SbO)C4H4O solution, 15
mL (NH4)6Mo7O24•4H2O, and 30 mL ascorbic acid solution (in that order) with stirring. Dispose after the
analysis is over.

F. Stock Phosphate solution (1.0 mL = 0.05 mg P)

Dry analytical grade KH2PO4 for one hour at 105oC. Cool and weigh exactly 0.2197 g. Transfer
quantitatively and dilute to 1000 mL in a volumetric flask.
Analytical Chemistry 17

G. Working Phosphate solution (1.0 mL = 0.5 g P)

Dilute 10.0 mL of the Stock Phosphate solution to 1000.0 mL.

H. Preparation of the Calibration Standards

Note: Rinse all volumetric flasks with nitric acid and distilled water. Any trace phosphate from the
detergents used will interfere with this analysis

Prepare nine (9) very clean 100.0 mL volumetric flasks. Prepare each flask according to the table below:

Flask number 1 2 3 4 5 6 7 8 9

Working Phosphate 0 1.0 3.0 5.0 10.0 20.0 30.0 50.0 0

solution (mL)

Sample (mL) 0 0 0 0 0 0 0 0 80.0

Coloring reagent (mL) 8 8 8 8 8 8 8 8 8

Dilute to volume with distilled water. Shake well and allow to stand for ten minutes. Read the absorbance
at 880 nm and 650 nm.
Analytical Chemistry 18

Some Spectrophotometric Methods

The following is a collection of classic methods in spectrophotometry that are considered as standard methods today.
These may be of importance to your course later on. If possible, the reference is the original publication of the
method.

Analyte Method Reference

Phosphorous (as Phosphate) Molybdate – Stannous chloride J.Murphy and J.P.Riley, Anal. Chim.
in water Acta., 1962, 27, 31-36.

Ammonia (NH3) in water Indophenol blue method (or L.Solorzano, Limnol. Oceanogr.,
Phenolhypochlorite method) 1969, 14(5), 799-801.

Iron (as Fe+2) in foods Ortho-Phenanthroline L.G. Saywell and B.B. Cunningham,
Ind. Eng. Chem., 1937, 9(2), 67-69.

Thiocyanate (SCN-1) in Separation by anion exchange P.Lundquist, J.Martensson, B.Sorbo

serum and urine followed by the Konig reaction and S.Ohman, Clin.Chem., 1979,
25(5), 678-681.

C.J.Vesey, H.McAllister and

R.M.Langford, J.Anal.Toxicol., 1999,
23(2), 134-135.

Thiocyanate (SCN-1) in urine Picrate Paper Method M.Rezaul Haque and J.Howard
Bradbury, Clin.Chem., 1999, 45(9),
1459-1464.

Nitrite (NO2-1) Griess Method J.B.Fox, Anal.Chem., 1979, 51(9),

1493-1502.
(An excellent review article).

Nitrate (NO3-1) Brucine Method D.Jenkins and L.Medsker,

Anal.Chem., 1964, 36(3), 610-612.
Analytical Chemistry 19

Properties of Buffer Solutions

When solutions of a conjugate acid and conjugate base are mixed, the resulting mixture is called a
buffer. Buffers have a specific pH depending on the amounts of the conjugate base and acid. The pH of a
buffer solution is computed by the Henderson – Hasselbach equation:

[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑏𝑎𝑠𝑒]
𝑝𝐻 = 𝑝𝐾𝑎 + 𝑙𝑜𝑔
[𝑐𝑜𝑛𝑗𝑢𝑔𝑎𝑡𝑒 𝑎𝑐𝑖𝑑]

Where the pKa is the pKa of the conjugate acid and the concentrations of the conjugate base and
conjugate acid are in molarity. It is worth noting that the equation explicitly shows that the pH is a
function of the ratio of the amounts of conjugate base and conjugate acid.

The objective of this experiment is to examine the behavior of a buffer solution when an acid, a base and
distilled water is added.

A. Preparation of Buffer Solutions

Prepare the buffer solutions according to the table below:

Label on CH3COOH NaCH3COO

Container 0.1 M 0.1 M

A 60 mL 0 mL

B 45 mL 15 mL

C 30 mL 30 mL

D 15 mL 45 mL

E 0 mL 60 mL

Mix the solutions well. Compute the theoretical pH for each solution. Measure the pH of
solutions A to E using a pH meter and record the pH following the table below:

Label on pH pH
Container (from pH meter) (from computation)

E
Analytical Chemistry 20

B. Effect of the addition of a strong base /acid and dilution with water.

Divide each buffer solutions in three 20 mL portions.

To the first portion, add 1 mL of 0.1M HCl. Mix well and record the pH.

To the second portion, add 1 mL 0.1M NaOH. Mix well and record the pH.

To the third portion, add 20 mL distilled water. Mix well and record the pH.

As a basis for comparison (i.e., control), prepare two 20 mL portions of distilled water. To the
first portion, add 1 mL 0.1M HCl. To the second portion, add 1 mL 0.1M NaOH. Record the pH.

Label on Initial pH 1st 20mL portion 2nd 20mL portion 3rd 20 mL portion
container (from pH meter) (+1mL 0.1M HCl) (+1mL 0.1M NaOH) (+20mL d. H2O)

d. H2O
(control)
Analytical Chemistry 21