Wollo University

College of Natural and Computational Science

Department of chemistry

Practical Instrumental analysis II Laboratory manual (Chem2054)

Prepared by: Binyam Abdu

Jan 2012

WOLLO UNIVERSITY PRACTICAL INSTRUMENTAL ANALAYSIS II LAB MANUALPage 1

Contents
Safety rules and protocols in the instrumental analysis laboratory ..................................... i
EXPERIMENT ONE ...........................................................................................................1
Determination of the refractive index of a methanol-ethanol solution ...............................1

EXPERIMENT TWO ........................................................................................................ 5

Refractive Index of a Liquid ............................................................................................... 5
EXPERIMENT THREE ......................................................................................................8
Determination of the concentration a given solution by Abbe Refractometer ...................8
EXPERIMENT FOUR ................................................................... Error! Bookmark not defined.
Determination of the percentage composition of the given mixture ................................ 10
EXPERIMENT FIVE ........................................................................................................12
Performance Characteristics of a Spectrophotometer .......................................................12
EXPERIMENT SIX ....................................................................................................... 21
Spectrophotometric Analysis of a Two-Component System with Overlapping Spectra .21
EXPERIMENT SEVEN ....................................................................................................24
Quantitative Analysis Using UV- Spectrometry ...............................................................24
EXPERIMENT EIGHT 29
Determining the composition of a two component system using spectrophotometer .....29

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Safety rules and protocols in the instrumental analysis laboratory
The following pages are intended to provide some general guidelines and mandatory behaviors for your
safety in our lab. These are sincerely meant primarily for your health and benefit. In addition, they are
also intended for your instructors’ peace of minds and the preservation of their professional reputations.
An instructor is responsible for your well-being in the laboratory; this responsibility is not taken lightly
by any intelligent professional, which is what all of you aspire to become.

Although a chemical laboratory is a dangerous place, it is probably safer than the average
household kitchen or, for that matter, trying to cross Rose Street. This is because there are
instructors around at all times who are concerned about your safety.

 Safety equipment and help is nearby, readily available, and even works.
 You are or will be aware of dangers and take proper precautions.
 People have spent many hours in designing safe laboratories, safe experiments, and
safety procedures.

Laboratory Signage: - When you first walk around the laboratory, note all the signage. The signage is
color-coded for your convenience:

 RED: - For the location of various Safety items such as showers and first aid kit, and for
disposal of Hazardous Waste.
 GREEN:-For the location of pure Reagents to be used in experiments.
 YELLOW:-For the location of miscellaneous items such as Instruments, Towel
Exchange, Drop Off and Pick Up of Unknown Containers.

Safety Glasses:-The Department of Chemistry requires that safety goggles must worn at all times in the
laboratory. This applies whether you are working at your bench, walking around, or waiting for a friend
to finish up his lab work. With permission safety glasses or case-hardened prescription glasses that
have side-shields may be born. If you wish to wait and not wear safety goggles, wait outside the
laboratory. Putting on your goggles is the first thing you do when you walk into the lab and the last
thing you do just before you leave. [Except, of course, for unlocking and locking your equipment
drawer

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Lab Coats or Aprons:-The Chemistry Department requires that students wear laboratory coats or
aprons when working in an instructional laboratory. Your normal lab equipment includes a rubber-
coated safety apron. There are a few white lab coats available for your use during the semester, and
there are also some disposable plastic-lined paper aprons available. If you’re interested, ask.

Lab Towels:-We cannot afford any longer to provide students a brand new cotton lab towel each
semester, but we have lots of used one’s for you. While your mother wouldn’t hang them in your home
bathroom or kitchen, they are usable. Whenever you want a new towel, drop your old one off in the
Used/Dirty towel box in the back of the room and pick up a New/Clean one. If you’ve just used your
towel to wipe up some concentrated acid or base, please rinse it out in a lot of cold water before you
throw it into the Used Towel box. Periodically, the dirty towels are washed, sterilized, dried, and
returned to the clean towel box. They may have holes in them and permanent spots, but they have been
washed and sterilized.

Safety Equipment:-Walk through the laboratory and learn the locations of all the safety equipment
well enough so that you can indicate their locations on a map of the laboratory. This is a possible exam
question. Know the quickest way to get to any of them from your assigned lab bench and from the
instrument area in the front part of the room.

Rubber Gloves: - If you wish to use protective gloves in the lab, the Department does provide latex
rubber gloves for your useon your hands. Don’t try to put one on your head or blow them up to make
balloons them look like cows’ udders. Their use is not mandatory. If your hands start feeling itchy or
you develop a pink or red inflammation on them, you may be allergic to latex. If you still want to use
gloves in the laboratory, tell a TA and we’ll order you some nonlatex gloves to use.

Accidents: - Report all accidents to a Teaching Assistant or whoever is in charge, no matter how minor,
and get first aid. We are obliged to report these to the University. In addition, having data on how
students get injured could well help us to clarify instructions or modify experiments or the lab itself.
Admittedly, it may be somewhat embarrassing to walk over to the TA with your finger wrapped in a
bloody paper towel; but please do not stand around with your hand in your pocket trying to finish up
the laboratory period, meanwhile bleeding down your leg and filling up your shoe. Don’t be
embarrassed to yell and/or scream for help if need be. You can’t die from shame, but you can die from
an accident.

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Unauthorized Experiments:-Absolutely no unauthorized experiments are permitted in the laboratory
at any time, for any reason. Punishment will be severe. This can include the involvement of campus
Security as well as the Dean of Students office.

Normal Laboratory Hours:-No laboratory work is allowed outside of the normal laboratory hours
without prior arrangement with a teaching assistant or a staff member each and every time. As a
supervisory person is required to be present at all times when a student is in the lab, laboratory work
outside the normal hours can only be scheduled at the convenience of an instructor.

Pipetting:- Do not mouth-pipette; use a rubber bulb. As a matter of fact, do not insert any laboratory
apparatus, glassware, supplies, or instrumentation into any of your bodily orifices. Although every
effort has been made to avoid or minimize the use of highly hazardous chemicals, you will still come
into contact with some fairly corrosive and/or toxic chemicals.

Pouring: - NEVER, NEVER pour reagent solutions above eye level. The container can slip out of your
hand, you can over-pour, you can miss completely, or the friendly buffoon at the next lab station can
bump your arm and leave you with a fanciful of reagent. In filling your burette, carefully place the ring
stand on which it is mounted on the floor or on one of the pull-out shelves. Alternatively, you can
remove the burette from the clamps, hold it below eye level and pour the solution in carefully. Use of a
small funnel helps to avoid spilling. If filled only with distilled or deionized water, you may use your
wash bottle to squirt down the inside of a burette mounted on the bench top.

Heat Burns:-Avoid burning yourself or others in the Chemistry laboratory. As there are no longer open
flames in the lab, about the only way this can happen is if you put some bodily part on a hot plate, grab
a flask or beaker containing a very hot solution, or remove something from a drying oven with your
bare hands.

Chemical Burns: - Immediately run copious amounts of cool water to the affected area. After
prolonged water-wash, you can remove the last traces with ordinary soap and water in the lavatory. The
affected area may need drying and a bandage. If you get any chemicals in the eye, you should head
right for an Eye Wash Station if you can. If you can’t, get to a water tap and call for help.

Cuts:- Small cuts are generally best rinsed in cold running water, allowed to bleed a bit to further clean
out the wound, dried with a sterile pad, and bandaged. Serious cuts are probably best treated by

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application of pressure with a cloth pad to the immediate area. Clean, preferably sterile, pads are
preferable.

Puncture Wounds:- All puncture wounds, such as spearing yourself with a glass or metal rod,
ramming a pipette end into your hand, or stepping on a nail are very dangerous because of the
possibility of tetanus. These will require further, professional medical care. Lockjaw is not very
pleasant.

“Pointing” Lab Equipment:-Never point the top of a test tube, beaker, flask, wash bottle, reagent
bottle, etc., yourself or anyone else. It may be loaded.

Jewelry:-Do not wear rings, watches, or other types of jewelry in the laboratory if at all possible. You
will be asked to remove what we might consider excessive amounts of hand and wrist jewelry. Such
items can catch on protruding objects, serve as nice traps for spilled chemicals keeping them in contact
with the body, function as nice “wires” to lead current into the body from electrical equipment resulting
in shocks and/or electrocution.

Fires:-Fires are highly unlikely in the Analytical Laboratory, but do avoid setting fire to yourself, other
students, instructors, or any parts of the laboratory. The laboratory is equipped with several fire
extinguishers. Learn where they are, and for what types of fires they are meant. Try to avoid fires, but
be prepared for one. Never use organic solvents when open flames are around. Do not use organic
solvents in those fume hoods that have an electric or gas hot plate or furnace? If necessary warn others
of fire by yelling “Fire!”… Loudly, really loudly

Electrical Equipment:-Anything, particularly aqueous electrolyte solutions, spilled around or on

electrical equipment can serve as a path for the conduction of lethal amounts of electrical current.

Ingestion:-Never put anything into your mouth or nose, or any other bodily orifice for that matter, in
the laboratory. Not even your finger. Food, drinks, drinking, or eating is strictly forbidden in the
laboratory. Go into the hallway for a snack or swig of “yuppie water” from a bottle. Smoking is not
permitted anywhere in the building by State law and by the University law. Take it outside. No tobacco
chewing or snuff; those are just plain disgusting.

Long Hair: - Loose long hair can be a danger. For example, bending over a burner can ignite it. Hair
spray, especially large amounts of a heavily lacquered type, can make hair extremely flammable. If you

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bend over, you can dip the ends into something caustic or toxic. Please try to keep your hair bound in
some manner.

Underwear: - Always wear clean underwear to lab. That way, if you have an accident and have to go
to the emergency room, the medical personnel can see that you wore clean underwear, and will no
doubt comment that you are wearing such nice clean underwear. In addition, your mother will be very
proud of you. All her years of nurturing and nagging have paid off.

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EXPERIMENT ONE

Determination of the refractive index of a methanol-ethanol solution

Objective: - To determine the composition of an unknown solution using its refractive index and the
calibration curve

Theory:

Refractive index

The speed of electromagnetic waves in vacuum, � ≅ 3 × 108 , is one of the most important constants in
chemistry. A human eye is able to detect electromagnetic waves in a range from 360 nm (violet color)
to 750 nm (red color). It is called a visible range of light.

When light waves travel through a medium (optical medium), its electric part interacts with the
electrons of that medium, causing them to vibrate. The electrons of the medium thus become radiating
light waves as the secondary sources. However, the speed of new waves, v, changes accordingly to the
optical properties of the particular medium.

It is always smaller than the speed of light in vacuum, V < C. All materials are characterized by their
ability to slow down the light waves, known as optical refractive index, n = -

The refractive index is a unit less parameter, equal to 1 for a vacuum and larger than 1 for any other
material (e.g. n=1.33 for water). The speed of light in air is only slightly less than C, resulting into the
refractive index of 1.0003. Typically, it is truncated to 1. The difference between a light speed in
different media results into the change of direction along which the light propagates, refraction (Fig. 1).
Refraction occurs when the light passes from one medium to a medium with a different index of
refraction, except the light that approaches the boundary between the two media perpendicularly.
Accordingly to the properties of an optical medium, some portion of light approaching the interface at
an incident angle α is reflected back to the first medium while the rest propagates into the other medium
at an angle of refraction β. The angles of incident, reflection and refraction are defined as angles
between the particular ray and the interface normal (see Fig. 1).

NB: the reflection angle is always equal to the incident angle.

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On the other hand, the refractive angle is determined by the Snell's law

�1��� � = �2 ��� �
Where n1 is the refractive index of medium 1 and

n2 is the refractive index of medium 2.

It is possible to define an optical density for the media of different refractive indices. Medium 1 has a
higher optical density than medium 2, if its refractive index is higher than that of medium 2. According
to the Snell's law, the light ray is "bending towards the normal" (β<α), if it enters the medium with a
higher optical density (Fig. 1). When it enters the medium with a lower optical density, it is "bending
away from the normal" (β>α).

Figure1. Refraction of light

Refractive index can be measured by the refractometer. We will use the double prism system called the
Abbe's refractometer, shown in Figure 2. It consists of the two optical prisms (illuminating and
refracting) with the thin layer of a liquid sample between them. The measuring prism is made of a glass
with a high refractive index (n2>1.75), which allows this refractometer to measure refractive indices up
to n1<1.75. The light enters the refractometer from the left side of the illuminating prism at many
different angles. The bottom part of this prism (AB') is rough, i.e. it consists of many small areas
oriented in different directions. As such, this surface can be imagined as a source shining the

Refractive index can be measured by the refractometer. We will use the double prism system called the
Abbe's refractometer, shown in Figure 2. It consists of the two optical prisms (illuminating and
WOLLO UNIVERSITY PRACTICAL INSTRUMENTAL ANALAYSIS CHEMISTRY II LAB MANUALPage 2
refracting) with the thin layer of a liquid sample between them. The measuring prism is made of a glass
with a high refractive index (n2>1.75), which allows this refractometer to measure refractive indices up
to n1<1.75. The light enters the refractometer from the left side of the illuminating prism at many
different angles. The bottom part of this prism (AB') is rough, i.e. it consists of many small areas
oriented in different directions. As such, this surface can be imagined as a source shining the light into
all directions. Part of this light passes through the sample into the refracting prism, where the biggest
possible angle of incident, α max, corresponds to the ray that propagates from point A to point B (Fig.
2). According to the Snell's law, the refraction of this ray is then described by the maximum angle of
refraction β max. All other rays enter the refracting prism at smaller angles and thus end up to the left
of point C. Consequently, detector located at the bottom of the refracting prism detects the illuminated
region to the left of point C and a dark region to the right of this point. Since the maximum angle, α
max, and the refractive index of the refracting prism, n2, is known constants, it is straightforward to
determine the refractive index of a measured liquid, n1.

The interface between an illuminated and dark region (position of point C) changes as a function of
angle β max, which is different for samples with different refractive indices n1. The simple readout
from the scale of refractometer then provides the refractive index directly, or it can be readily
determined using a conversion table.

Fig. 2 The schematic of the Abbe's refractometer

The determination of the refractive index of a methanol-ethanol solution

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The task consists of the determination of the refractive index of methanol (CH3OH), ethanol
(CH3CH2OH) and their mixtures described by the volumetric percentage of methanol (V% CH3 OH).
The measurement results into calibration curve (refractive index as a function of V% CH3OH). This
curve will be then used to determine the composition of an unknown mixture of methanol and ethanol.

Apparatuses

 Abbe's Refractometer
 Pipette
 Thermometer
 Filter Paper

Chemicals:

 Different mixtures of methanol and ethanol at known compositions, mixture of methanol and
ethanol at an unknown composition,

Procedure:

1. Turn on the light source.

2. Open the double-prism of the refractometer, clean both glass surfaces with a filter paper, and close
the double-prism. Use a pipette to fill the space between the two prisms with methanol. Turn the
refractometer scale knob to get a clear interface between the illuminated and dark regions. Use the
micrometric screw for the additional refinement of the scale, until the clear interface appears. Read
out the integer value from the rough scale and decimal number from the refined scale. Determine
the index of refraction using the conversion table.
3. Open the double-prism and dry out glass surfaces using the filter paper. Repeat the measurement
for all methanol/ethanol mixtures of known and unknown compositions.
4. Use MS Excel to create a calibration curve (i.e. dependence n=f (v% CH3OH)). Fit the
experimental points with a straight line and write down its coefficients.
Determine the composition of an unknown solution using its refractive index and the calibration curve

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EXPERIMENT TWO

Refractive Index of a Liquid

Objective: To determination of Specific and Molar refractivity of some solutions

Theory: The Refractive Index (R.I.) is an important property of the molecular structure of a liquid. For
pure hydrocarbons, one can get an idea of aromatic content of a liquid using R.I.

Table

Sn Liquid Refractive Index

1 aromatics 1.50

2 Naphthene 1.44

3 saturates 1.37

When a light beam passes from one substance into another, the beam is refracted (bent) so that it
travels in a different direction. The extent of refraction depends on:

 The relative concentration of atoms or molecules

 The structure of the atoms or molecules

A refractometer measures this property (R.I.) using the D line of sodium.

Hence , and n is determined from angle i and N, then R.I. is reported as nD_________

The R.I. is measured by the critical angle method with a Bausch and Lomb or Abbe Refractometer
using monochromatic light.

In the Abbe Refractometer liquid is placed between two prisms, which are rotated to obtain grazing
refraction angle. This is achieved by a focusing lens, which gives a dark field and a light field in the
view piece. When the two fields are equal (as determined by a set of crosshairs), the angle is measured.
A calibrated scale gives the R.I. directly for this angle and the R.I. of the prism.

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Molar Refraction, Mr

Refraction of light is an additive property, but also depends upon the structural arrangement of

atoms in a molecule. From the electromagnetic theory of light, the molar refraction, Mr, is given by:

The molar refraction can be calculated from the sum of the individual atomic refractions of
the atoms and groups present. This can sometimes be used to determine the structure of an
unknown compound whose molecular formula is known. It is not useful for mixtures.

Chemicals:

 2-proponol
 Ethylene glycol
 Acetone

Apparatuses

 Abbe's Refractometer
 Pipette
 Thermometer
 Dropper

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Procedure:

1. Clean the surface of the prism first with alcohol and allow it to dry. Set the bath temperature to
20 °C.
2. Open the prism and place approximately two drops of the liquid sample on the top of the bottom
prism. Use care not to touch the prism surface with the dropper. Common samples are 2-proponol
and ethylene glycol. Check with lab instructor.
3. Close the prism case and place the lamp close to the prism.
4. Turn on the lamp.
5. Adjust the hand wheel on the right side of the instrument until the viewer appears light above and
dark below the center horizontal line.
6. Adjust the compensator dial on the front until the image in the viewer shows a sharp line between
light and dark.
7. Adjust the hand wheel on the right until the horizontal line in the viewer passes between the
crosshairs.
8. Depress and hold the switch on the left and read the refractive index value. Then take three set of
readings and find average of three readings.
9. Clean the prism with acetone and a Kim wipe.

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EXPERIMENT THREE

Determination of the concentration a given solution by Abbe Refractometer

Objective: To determine the concentration of the given potassium chloride solution using Abbe
Refractometer.

Theory

The speed of light in vacuum is always the same, but when the light moves through any other
medium it travels more slowly since it is constantly being observed and reemitted by the atom
in the material.

Since density of liquid usually decreases with temperature, it is not surprising that the speed of
light in a liquid will normally increases as the temperature increases. Thus the index of
refraction normally decreases as the temperature increases from a liquid. ƍ =

Chemicals


20% of potassium chloride solution

Distilled water

Acetone
Apparatuses


Volumetric flasks

Abbe Refractometer

cotton
Procedure:

1. Prepare 20% of potassium chloride solution in a 50 mL standard flask using distilled water, from
this solution prepare 15% , 10% , and 1% potassium chloride solution in a 50 mL standard flask by
way of dilution.
2. Place the Abbe Refractometer on a table preferably near a window. Turn the latch and release the
lower prism.
3. Clean the prism gently with cotton moistened with alcohol. Put a drop of 1% potassium chloride
solution on the glass surface and close the prism with the help of the latch.

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 4. Adjust the mirror in such a way that maximum light enters the window prism. Then rotate the
prism with the help of movable arm until the border line appears in the field.
5. Adjust for sharp boundary line and note down the refractive index reading. Then open the latch
and clean the prisms.
6. Take a sample from the 5% potassium chloride solution and determine the refractive index for all
the solutions and tabulate them.
7. Draw a calibration plot by taking the refractive index in the ‘Y’ axis and concentration ‘X’ axis.
Find out the refractive index of the unknown solution and then determine the concentration from
the calibration chart.

Result: the concentration of the given potassium chloride solution: _______________

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EXPERIMENT FOUR

Determination of the percentage composition of the given mixture

Objective: To find out the percentage composition of the given mixture of NaCl and NaOH.

Theory:

For the titration of a solution of NaOH and NaCl the other solution need is HCl solution. NaOH react
with HCl solution whereas NaCl will remain as such.

NaOH + HCl NaCl + H2O

The mixture solution will be titrated with the acid solution of HCl and we can find out the
normality and strength of NaOH. By dividing the strength of NaOH from the total known strength
of the solution we will find out the strength of NaCl and hence find the % composition.

Chemicals


N HCl Sodium Hydroxide

Sodium chloride

Phenolphthalein Indicator
Apparatus


Burette

Pipette

Conical Flasks

Beakers
Procedure:

1. Rinse the burette with the given N/10 HCl solution

2. Take the HCl in the burette and note the initial reading
3. Pipette out the 10 mL of the solution in the conical flask
4. Add a drop of indicator in the solution
5. Add the acid solution from burette in to the conical flask till solution become colorless

6. Note the burette reading

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 7. Repeat the experiment to get three concordant reading
Observations:

Normality of the HCl solution = 0.1 N

Volume of the mixture of the in conical flask= 10 mL

Table

Sn Initial Readings Final Readings Volume 0.1 N HCl used (ml)

1
2
3
Concordant Reading =_________ mL
Calculations:

By the law of chemical equivalents N1V1 = N2V2

Normality of given NaOH solution

As the equivalent weight of NaOH = 40

� �
Strength of given NaOH solution = �2 × 40 � = �( �)

Strength of given NaOH solution = N2x 40 gm/l = y….. g/l

Because the amount of NaOH in the mixture is y g/L

Amount of NaCl in the mixture = (8 – y) g/L = z... g/L

The percentage of the NaOH = y/8 x 100,

The percentage of NaCl =

Questions:

1. What do you mean by back- titration?

2. What is the colour of phenolphthalein in acidic and base medium?
3. What is the structure of phenolphthalein?

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4. What is the procedure to prepare a 0.1 N solution of NaOH?

EXPERIMENT FIVE

Performance Characteristics of a Spectrophotometer

Objective: To investigate several characteristics of a commercial spectrophotometer and compare
different cuvette materials

Theory

Spectroscopy In this experiment you will become familiar with different features of
commercial ultraviolet-visible (UV/vis) spectrophotometers. The prefixes ultra and infra mean
above and below, respectively. Hence, the energy of ultraviolet radiation lies just above the
visible violet light (i.e., ultraviolet means above visible violet light in energy). Likewise,
infrared means below visible red light in energy. Because the energy range for a typical UV
analysis is right next to the energy range of visible light, many instruments, including ours,
operate in both the UV and visible regions.

In this lab period, you will learn how to prepare samples for analysis and how to obtain data
from the UV-Vis instrument. The samples are compounds that might be found in explosives.
They are nitro-aromatics or compounds that contain nitro groups (-NO2) bonded to an aromatic
ring. In this case, the aromatic ring is toluene. You will examine the output of the instrument in
the wavelength region between 200 and 900 nm (nanometers). One nanometer is 1x 10-9meter.

Background Theory for UV Absorption in terms of energy, the IR region lies below the visible
region, which lies below the UV region. Thus, IR is strong enough to cause atoms within a
molecule to vibrate, but not strong enough to cause electrons to change orbital locations. UV
radiation between 200 and 400 nm is strong enough to cause loosely held electrons to change
locations. These electrons can be either the non-bonding electrons (n-electrons) of aldehydes
or ketones, or they can be the π-electrons of conjugated π- systems.

Ultraviolet spectroscopy is one of several forms of spectroscopy that we will study this semester.
Accordingly, it is important that you understand the capabilities and limitations of each of these forms
of spectroscopy. The words spectroscopy and spectrometry have different meanings. A spectroscope is

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an instrument, and spectroscopy is the use of a spectroscope. Spectrometry means the measurement of
a spectrum. One generally measures wavelengths or frequencies, or a spectrum of them. We will use
the electromagnetic spectrum to gain information about organic molecules.

The modifier ultraviolet means that the information will come from a specific region of the
electromagnetic spectrum called the ultraviolet region. The electromagnetic spectrum includes all
radiation that travels at the speed of light c (3 x 1010 cm/sec). The electromagnetic spectrum includes
radio waves, which have long wavelengths, x-rays, which have short wavelengths, and visible light,
which has wavelengths between those of radio waves and x-rays. All of these waves travel at the speed
of light. We normally describe these waves in terms of their energy. Of the three kinds mentioned, x-
rays are most energetic, visible light next, and radio waves least energetic. Thus, the shorter the
wavelength is, the greater the energy of an electromagnetic wave.

Electromagnetic radiation (EMR) has a dual nature; it has the characteristics of both waves and
particles. Both forms of EMR are important. From the wave nature of the waves we get the wavelength
(λ) or distance between two crests. The wavelength is related to the frequency (ν), how many
wavelengths pass a given point in a given time, by the velocity of the wave c. From the particulate
nature of EMR, we get the energy E of a given wave, which is proportional to its frequency. Plank’s
constant h turns the proportionality into an equation. The mathematical relationships among these
variables are shown below.


Frequency and Wavelength: ν = c/λ or λ = c/ν or c = νλ

Energy and Frequency: E α ν or E = hν

Energy and Wavelength: E = hc/λ
Visible light includes the rainbow colors red, orange, yellow, green, blue, indigo, and violet. A handy
acronym for these colors is ROYGBIV, which most people remember from grade school. Note red is at
the low-energy end of the visible spectrum and violet is at the high-energy end. These facts allow us to
quickly understand the terms infrared and ultraviolet. The prefix infra means below, and the prefix ultra
means above. Thus, infrared radiation is outside the visible range and lies just below red on the energy
scale. That is, infrared radiation is less energetic than visible light. Ultraviolet radiation is outside the
visible range and is just above violet on the energy scale. Thus, infrared literally means “below” red (in
terms of energy), and ultraviolet means “above” violet (in terms of energy). The UV energy of the sun
is what gives ride to sunburns.

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 Figure 3 Electromagnetic radiations (EMR)

We learned in general chemistry that visible yellow light is observed when sodium ions are heated in a
Bunsen burner. The heat excites some ground-state electrons to higher energy levels, then when the
electrons “fall” back to the ground state, they “emit” energy that corresponds to the energy difference
between the energy states (orbitals) where the electrons are found. When this energy difference falls
within the energy range of visible light, we can see it as a color. In the case of sodium, we see yellow
light. Thus, the light results from the emission of energy by the electrons as they fall from higher t o
lower energy states (orbitals). Note that it takes the same amount of energy to make the electrons jump
from the lower to higher states as the amount of energy the electrons emit when they fall from higher to
lower states. We generally add more energy than is absolutely necessary for the transition to ensure that
the transition occurs. When we add energy to a system, we give it a positive sign (endoenergetic).

When a system gives off energy, we give it a negative sign (exoenergetic).Just as heat causes some of
sodium’s electrons to move to higher energy states, ultraviolet radiation causes electrons in certain
organic compounds to move from their ground state locations to orbitals of higher energy.

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The Instrument

You will perform checks on the following performance characteristics


Wavelength calibration: - How accurate are the reported wavelengths?

Linearity:-Is the photometric response linear over a reasonable absorption range?

Resolution: - How small of a wavelength difference can be detected?

Effect of Slit Width: - How does the exit slit of the monochromator affect the absorption
spectrum?
Each of these is discussed in further detail below.

Wavelength Accuracy

The wavelength accuracy of a spectrophotometer is the correctness with which the wavelength
of light reaching the sample matches the wavelength reported by the instrument. When a
wavelength is selected, the monochromator’s grating (Grating 2 in Figure IB-1) is rotated so
that the specified wavelength is centered on the exit slit of the monochromator. Errors can
come from two sources.

First, imperfections in the positioning system might cause errors in the grating rotation, which
in turn causes errors in the wavelength reaching the sample. Second, the light reaching the
sample is not a single wavelength, but rather a band of wavelengths. The width of this band is
the spectral bandwidth (SBW) of the instrument, and is defined as the width in nanometers of
the band of light leaving the monochromator measured halfway between the baseline and the
peak intensity. Wavelength errors tend to increase with SBW. Wavelength accuracy is verified
using calibration standards. In general the two standards that are used are the D2 source lamp
lines and a holmium oxide (Ho2O3) filter. Both are relatively stable and have several well
defined peaks for calibration purposes. In this experiment we will use the holmium oxide filter.

Linearity

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Photometric linearity is the ability of the optical detector to generate a change in electrical output that
is proportional to a change in incident light intensity. The detector must exhibit photometric linearity
over the entire operating wavelength range of the spectrophotometer and over a range of light
intensities.

Photometric linearity will be assessed using a series of standards of either Ni2+, Co2+, or Cu2+. (Of
course it is important to realize that the solutions must be made correctly in order to truly assess the
spectrophotometer’s linearity. Errors in solution preparation will appear as instrumental errors.)

Resolution and the Effect of Spectral Bandwidth

Resolution refers to the extent to which two peaks or bands (e.g., spectral, chromatographic, etc.) can
be separated spatially such that peak overlap is minimized. The limiting resolution of the
spectrophotometer depends upon the narrowest SBW that can be achieved. For high resolution work a
very narrow SBW is required. A solution of toluene in hexane will be used to assess the resolution of
the instrument. The SBW of an instrument is a function of the exit slit width (w) and the reciprocal
linear dispersion (D-1) of the monochromator. For a given value of D-1, the SBW is given by, SBW =
wD−1

The reciprocal linear dispersion of a spectrophotometer is usually fixed, so SBW is controlled by

changing the slit width. Using the spectrometer manual, determine D-1 in nm/mm and SBW range in
nm.

Smaller values of SBW (i.e. a smaller slit width) produce higher resolution spectra. But because the
light intensity reaching the sample also decreases with slit width, spectra recorded with a small SBW
also exhibit decreased signal-to-noise ratios. Conversely, larger values of SBW produce spectra with
less noise (more light reaches the sample) but also with less resolution. The effect of SBW on the
absorption spectrum of benzene vapor will be investigated as part of this experiment.

Safety Considerations

 Benzene is a carcinogen. Do not open the cuvettes containing the benzene vapor.
 Hexane and toluene are flammable and toxic.
 Dispose of hexane, toluene in the appropriate waste container.
Chemicals

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 
0.020 %v/v Toluene in Hexane

20-500 mM Ni2+, Co2+, or Cu2+ solutions

Cyclohexanone

Benzene

distilled water
Apparatuses


Quartz cuvettes

Holmium Glass Filter

UV Spectrophotometer,

Volumetric Flask,

Pipettes and

Beakers
Procedure


This experimental procedure has several different parts, but they do not need to be done in
sequentially
NOTE: This procedure does NOT need to be performed every time you use the
spectrophotometer. However, it is important to realize that just because the readout from the
instrument indicated that 500.00-nm light is being measured, this does not absolutely guarantee
that this is the case. If wavelength accuracy is of primary concern, then you should verify the
spectrophotometer’s calibration by measurement of the absorption lines of holmium glass or
emission lines of the deuterium (D 2) lamp.


Before using the instrument, prepare the following solutions. (Note that some of these will be
provided for you; check with your instructor)
i 0.020 %v/v Toluene in Hexane (UV grade)
ii ii. 20-500 mM Ni2+, Co2+, or Cu2+ solutions

Have appropriate blanks ready for each solution

Use quartz cuvettes for all components,
unless otherwise noted.

1. Wavelength Calibration and Resolution

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Holmium Glass Method: - This method allows you to verify the wavelength calibration at
three different wavelengths, 460.0 nm, 360.9 nm and 279.4 nm. The performance is
considered satisfactory if the wavelength errors of the holmium glass absorption lines are
within ±1 0.3 nm

(a) With the Holmium Glass Filter in the sample compartment, record the spectrum between
250 and 500 nm. at a 0.5 nm bandwidth, 10 nm/min scan speed, and a data interval of 0.1 nm.
(b) Determine the wavelengths of maximum absorbance using the Peak Pick Table. How does
the spectrum compare with the \wavelength values reported above?
Resolution: Record the spectrum of the toluene/hexane solution over the full range of the
instrument for a range of slit widths low to high. At what point can the “fine structure” no
longer be seen? How is this related to the resolution of the instrument?

2. Effect of slit width

Place one drop of benzene in the bottom of a clean dry quartz cell and seal the cell. Do not get
benzene on the walls of the cell. Put cell in the sample compartment; no reference is necessary.

Set up the instrument to scan between 300 and 200 nm using a data interval of 0.200 nm (the
scan rate should automatically adjust to 120 nm/min). Record the absorption spectrum of
benzene vapor in the provided cuvette at spectral band widths (SBW) of 0.2, 0.5, 1.0, and 2.0
nm. Also record the spectrum using the Ocean Optics diode array spectrophotometer.

Q1. What is the effect of spectral bandwidth on the absorption spectrum of benzene? Comment
in terms of resolution and in terms of signal-to-noise ratio.

Q2. Estimate the SBW for the diode array spectrophotometer by comparing the benzene spectrum to
the spectra recorded at different SBW on the Unicam.

Q3. When might a small SBW be necessary? When might a small SBW be a disadvantage?

3. Absorption properties of cuvette materials

On a single graph, overlay spectra (recorded with the Unicam instrument over the entire uv-
vis range) of cuvettes made of glass, plastic, and quartz. Leave the reference cuvet empty
when recording this spectrum and use a reasonably fast scan rate (~700 nm/min). Also, be
sure to record a blank spectrum (i.e. no cuvette in either holder). Include the overlaid spectra
in the report.
WOLLO UNIVERSITY PRACTICAL INSTRUMENTAL ANALAYSIS CHEMISTRY II LAB MANUALPage 18
 4. Linearity
Find the λ max of Ni2+, Co2+, or Cu2+ (whichever ion you selected for preparing the set of 5
solutions). Record the absorbance of each solution at the λmax you determined. Make at least
3 measurements of absorbance for each solution. Each time you make a replicate
measurement, be sure to remove and replace the cuvette. (This will help to account for
positioning errors.)

Q4. Does the spectrophotometer respond linearly to concentration? Explain how you determined
this and how you can quantify the linearity of the response.

Nitro aromatics

a) Place the reference and sample cuvettes in the instrument in the correct locations.
b) Obtain the spectrum from 200-900 nm and save the data points of the spectrum. Note that
our instrument scans both visible and ultraviolet regions of the electromagnetic spectrum.
c) Repeat the procedure for using the other solutions of the nitro aromatic (in three solvents total).
For each of your nitro aromatic solutions, write out the appropriate UV data in the format
you would expect to see in a scientific journal (e.g., nitro aromatic: λmax 250 nm
(ethanol), ε 12,000).

Determine whether the change in solvent resulted in a bathochromic, hypsochromic or no

shift for the nitro aromatic compound. (Show all spectra on the same graph, and clearly label).
What conclusion(s) can you draw from your data about the nature of the transitions in the
compound?

Also to be include in the Report:

1. The report should contain a schematic diagram of the internal workings of the instrument.
2. For what types of molecules is UV-VIS spectroscopy most useful? For which is it not? As a
consequence, what are the best solvents for UV-VIS spectroscopy?
3. What is meant by bandwidth? As you narrow the bandwidth of the UV-VIS spectrometer, what
might you expect to happen to the spectrum?
4. Labeled copies of all the spectra and peak data.
5. Discuss in what situations would an accurate calibration of the UV-VIS be most important?
6. Discuss how changing the bandwidth changes the spectrum of the benzene vapor.

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7. Compare the different optical material used to make cuvettes. What wavelength ranges is each
material useful for?
8. Explain what is meant by lambda max (λmax).

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EXPERIMENT SIX

Spectrophotometric Analysis of a Two-Component System with Overlapping

Spectra
Objective

 To calculates the concentration of the components of the mixture

 To determine composition of a two-component system using spectrophotometer
methods.

Theory

A number of methods have been developed to determine the composition of a binary mixture
spectrophotometrically. Most of these are directed at mixtures where one component can be
isolated from the other or they require a Beer’s law experiment to measure the molar
absorptivity of each of the substances in the mixture. However, Blanco, et. al. described a
method of resolving mixtures with overlapping spectra, called MultiWavelength Linear
Regression Analysis or MLRA, without determining molar absorptivity or complicated
mathematics. Using Blanco’s method, the composition of a binary mixture with overlapping
spectra can be resolved with only three measurements, the absorbance of a standard solution
for each component, and the unknown mixture itself.

Here’s how MLRA works: Assuming additivity, the absorbance of a mixture is the sum of the
absorbencies of its components. If we have a mixture consisting of two components, 1 and 2,
with an unknown concentration of C1 and C2, then: Absorbance of the unknown mixture, A
mixture = A1 + A2 but applying Beer’s law: A1 = ε 1 b C1 and A2 = ε 2 b C2. Substituting: A
mixture e= ε1 b C1 + ε2 b C2. However, the absorbencies of standard solutions of the same
substances will follow the same Beer’s law relationship and have the same molar absorbance,
ε , and one centimeter path length, b, as the unknown solutions under the same conditions.
Therefore, we can write:

A standard 1 = ε1b C standard 1 and A standard 2 = ε2b C standard 2

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 Dividing by A standard 1 and simplifying we obtain

Therefore, a plot of will give a slope of and y intercept of . That is, the concentration of the
unknown component in the mixture 2, equals the slope times the concentration of the standard
solution for component 2. Likewise, the concentration of the unknown component in the mixture 1,
equals the product of the intercept times the concentration of the standard solution for component
1, or simply C 2= m Cstandard 2 and C 1= b Cstandard 1

Let’s apply this method to the analysis of the metals in a United States five-cent coin, A.K.A.,
a nickel. To this end, the absorption spectra of the standard solutions with known
concentrations and that of the coin are obtained as three overlapping curves on a single graph.

Chemicals


Nickel and Copper Wire

Nitric acid

Deionized water
Apparatuses


Uv - Spectrophotometer,

Cuvettes,

Volumetric Flask,

Pipettes

Beakers

Procedure
WOLLO UNIVERSITY PRACTICAL INSTRUMENTAL ANALAYSIS CHEMISTRY II LAB MANUALPage 22
1. Prepare solutions of the standards by dissolving 0.5 grams of nickel wire in dissolved in 15 mL
of 1:1 nitric acid and diluted to 50 mL with deionized water. The copper standard solution is
prepared similarly, by dissolving of 0.2 grams of copper in 5 mL of the 1:1 nitric acid solution and
again, diluted to 50 mL with deionized water. The U.S. five cent coin solution was prepared by
dissolving 0.13 grams of a nickel 5 cent coin in 5 mL of the same nitric acid solution and diluted to
50 mL.
2. The spectrophotometer, calibrated using a distilled water blank, is used to measure the absorbance
vs. wavelength for each of the three solutions prepared above.
3. Now that the absorbance of each solution has been measured, remove from consideration
absorbance values which are the most unreliable (those at the extreme ends of the spectra or those
outside the optimum 0.15 - 0.70 absorbance range).
4. Use the procedure described above and, calculates the concentration of the components of the
mixture using the slope and intercept of the line of best fit. Use this information to determine the
percentage composition of the metals in a U.S. five-cent coin based on the MLRA.
5. Be sure to include quantitative error analyses of your results. Compare your results with the
composition stated by the US mint.

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EXPERIMENT SEVEN

Quantitative Analysis Using UV- Spectrometry

Objective: To introduce the principles of UV-spectrophotometer as well as the operating
characteristics and apply Ultraviolet-visible spectrometry for quantitative measurements.

Theory:

Ultraviolet-visible Spectrometry is based upon absorption of electromagnetic radiation in the visible

and ultraviolet regions of the spectrum resulting in changes in the electronic structure of ions and
molecules. When ions or molecules are exposed to light having an energy that matches a possible
electronic transition or charge transfer energy, some of the light energy will be absorbed as the
electron is promoted to a higher energy orbital or as a charge is transferred from donor atom to
accepter atom within the ion or molecule.

Electronic transition: The valence electrons in organic molecules, and inorganic anions such as
CO32–, occupy quantized sigma bonding, σ, pi bonding, , and nonbonding, n, molecular orbitals.
Unoccupied sigma antibonding, σ*, and pi antibonding, *, molecular orbitals often lie close enough
in energy that the transition of an electron from an occupied to an unoccupied orbital is possible. The
possible transitions include:


→ * and → * transitions: high-energy, accessible in vacuum UV (λmax <150 nm). Not usually
observed in molecular UV-Vis.

n → * and → ∗ transitions: non-bonding electrons (lone pairs), wavelength (λmax) in the 150-
250 nm region.


n → * and → * transitions: most common transitions observed in organic molecular
UV-Vis, observed in compounds with lone pairs and multiple bonds with λmax = 200-600 nm. Of
these transitions, the most important are the n → * and → *, because they involve functional groups,
or double/triple bonds that are characteristic of the analyte and wavelengths (Uv/vis) that are easily
accessible.

Many transition metal ions, such as Cu2+ and Co2+, form solutions that are colored because the metal
ion absorbs visible light. The transitions giving rise to this absorption are due to valence electrons in
the metal ion’s d-orbitals. For a free metal ion, the five d-orbitals are of equal energy. In the presence

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of a complexing ligand or solvent molecule, however, the d-orbitals split into two or more groups that
differ in energy.

Charge transfer: Many inorganic and organic complexes exhibit this type of absorption and are
therefore called charge-transfer complexes. A charge-transfer complex consists of an electron donor
group bonded to an electron acceptor. When this product absorbs radiation, an electron from the
donor is transferred to an orbital that is largely associated with the acceptor. For example the
ferro/ferricyanide complex responsible for the color of Prussian blue. The red color of the iron
(llI)/thiocyanate complex is a further example of charge-transfer absorption.

Instrumentation: UV/Vis spectrometers consist of five basic components, (i) a light source (ii) a
mechanism to select a specific wavelength of light in the UV/visible region of the spectrum,

(iii) a chamber where a cuvet containing a test solution can be introduced into the light path, and (iv)
a photocell that can determine the amount of light absorbed by the sample (or the intensity of light
transmitted through the sample), and (v) signal processor and readout unit.

On most spectrophotometers two scales are present, %T and A. For a uniform absorbing
medium (solution: solvent and solute molecules that absorb light) the proportion of light
radiation passing through it is called the transmittance (T) and the proportion of light
absorbed by molecules in the medium is absorbance (A). Transmittance of a sample is
defined as:

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 T=

Where Io is intensity of the incident radiation entering the medium and I is intensity of the
transmitted radiation leaving the medium. Transmittance is usually expressed as percent
transmittance (%T):

% T= 100

The relationship between transmittance (T) or percent transmittance (%T) and absorbance (A)
is given by the following equation:

A= log( )=2-log(%T)

Absorbance has no units (Why?) and varies from 0 to 2 (linear region for most
substances is from 0.05 to 0.7). The Beer-Lambert Law states that absorbance (A) is
proportional to the concentration

(c) of the absorbing molecules, the length of light-path through the medium and the molar
extinction coefficient:

A= εcl

Where ε is molar extinction coefficient for the absorbing material at wavelength in units of
1/(mol x cm), C is concentration of the absorbing solution (molar) and l is light path in the
absorbing material (l = 1 cm for our purposes).

The absorption spectrum (plural, spectra), or more correctly the absolute absorption spectrum,
of a compound may be shown as a plot of the light absorbed by that compound against
wavelength. Such a plot for a colored compound will have one or more absorption maxima
(λmax’s) in the visible region of the spectrum (400 to 700 nm). Λmax is the wavelength at
which maximum absorption in the spectrum occurs. To obtain an absorption spectrum, the
absorbance of a substance must be measured at a series of wavelengths. Absorption in the
visible and ultraviolet regions can be measured by UV/visible spectrophotometer.
WOLLO UNIVERSITY PRACTICAL INSTRUMENTAL ANALAYSIS II LAB MANUALPage 26
 The use of visible and UV spectrometry for quantitative analysis by comparing the
absorbance of standards and samples at a selected wavelength is one of the most widespread
of all analytical techniques. It is also one of the most sensitive. The analysis of mixtures of
two or more components is facilitated by the additivity of absorbance’s that will be discussed
in next experiment. Other applications include measurement of the absorption of complexes
as a function of solution conditions or time to establish their composition, and to determine
thermodynamic and kinetic stability for analytical purposes or for more fundamental studies.

Apparatuses


UV Spectrophotometer

Cuvette

volumetric flask

Pipettes

Beakers
Chemicals

 0.25M stock solution of CoCl2, 0.5M stock solution of Ni(NO3)2,

 0.1880M cobalt (II) chloride solution
 Distilled water.

Procedure:

A. Determination of Maximum Wavelength (λmax)

1. Prepare the following solutions
a) 0.1M CoCl2, by pipetting 10ml of the 0.25 M stock solution of CoCl2 into a 25 mL
volumetric flask and diluting to the mark and mix well.
b) 0.2M Ni(NO3)2, by pipetting 10ml of the 0.5 M stock solution of Ni(NO3)2 into a 25 mL
volumetric flask and diluting to the mark and mix well.
2. Obtain four matched cuvettes. Set the wavelength and adjust the instrument to read A = 2
(0%T) with no cuvette and A = 0 (100% T) when the water (the solvent) filled cuvette in
the sample holder.

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 3. Remove the water (Solvent) filled cuvette and insert approximately 3ml of sample tube;
read the absorbance.
4. Measure the absorbance of 0.1M CoCl2 and 0.2M Ni(NO3)2 solutions at wavelength
intervals of 20 nm as follows.
 With cobalt chloride, start at 600 nm and work downwards.
 With nickel nitrate, start at 750 nm and work downwards.
5. Plot the absorbance versus the wavelength for the two solutions on different graph papers.
6. Determine maximum wavelengths (λmax) of the two solutions.
B. Quantitative Analysis
1. Obtain approximately 75 mL of 0.1880 M CoCl2 stock solution in a small beaker. Make
solutions that are 0.0376, 0.0752, 0.1128, and 0.1504 M concentration of cobalt solution
in 25 mL of volumetric flask.
2. Adjust the wavelength to the wavelength of maximum absorbance of CoCl2.
3. Adjust the spectroscopy from 0 and 100% T, using the same cuvette for distilled water
blank.
4. Remove the cuvette from the sample holder and see if the meter returns to zero, continue
this until 0 and 100 are obtained with the cell out and in, respectively.
5. Measure the absorbance of the four known concentration solutions; with each successively
more concentrated solution, at the selected wavelength.
6. Obtain an unknown solution and determine %T and the absorbance A of this solution at
the same cuvette and wavelength selected previously.
7. Plot log %T verses concentration and absorbance verses concentration for the set of data
obtained at the fixed wavelength. (A= 2- log %T).
8. Find the concentration for the unknown solution

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EXPERIMENT EIGHT

Determining the composition of a two component system using

spectrophotometer
Objective: To quantitatively determine the composition of a two-component system using
spectrophotometer methods.

Theory:

It is possible to analyze a complex mixture containing several species simultaneously

without prior separation. According to beer’s law, the absorbance (a) of a solution is
equal to the absorbtivity (ε) times the cell length (b) times the concentration (c) at a
given wavelength or:

A = εbc since b is usually one cm, the working equation reduces to A = εc.

The beer’s law also applies to solutions containing more than one kind of absorbing
substances. Provided that there is no interaction among the various species, the total
absorbance for multi component systems is the sum of the individual absorbance. In
other words:

A total = A1 +A2 +…+ An= ε1bc1 + ε2bc2 + …+ εnbcn

Where the subscripts refer to absorbing components M,N,…n.

To analyze a solution containing a mixture of species 1 & 2 (binary mixture), molar

absorptivity of for the individual M & N species are first determined at wavelengths λ1
& λ2 using standard solutions. To complete the analysis, the absorbance of the mixture
is determined at the same two wavelengths. From the known molar absorptivity’s and
path length, the following simultaneous equations hold:

A1 = εM1bcM1 + εN1bcN1

A2 = εM2bcM2 + εN2bcN2

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 By determining the absorptivity’s (ε) of each species at each wavelength, we can
theoretically determine the concentration of each component in a mixture by measuring the
total absorbance at each wavelength and solving the resulting equations simultaneously.

Apparatus:

 Uv –Spectrophotometer
 Cuvettes
 Volumetric Flask
 Pipettes
 Beakers.

Chemicals:

 Cobalt solution
 Nickel solution

Procedure:

1. Make sure the instrument is zeroed at the nickel λmax.

2. At the nickel λmax (λ1), determine % transmittance and/or the absorbance of the known
nickel and known cobalt.
3. Re-zero the machine at the cobalt λmax 510 nm. (λ2).
4. At the cobalt λmax, measure the known nickel and the known cobalt as above.
5. Empty the cuvettes containing known nickel and cobalt solutions and refill them (with
correct rinsing) with your unknown cobalt. Also fill another cuvette with the unknown
mixture.
6. Since the instrument is still at the cobalt λmax (λ2), re-zero, and measure the unknown
cobalt and unknown mixture at the cobalt λmax. (Be sure to put your data in the correct
table).
7. At the nickel λmax, zero the instrument.

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 Data Table:

Concentration of known nickel solution

Concentration of known Cobalt solution

Absorbance of known nickel solution Co λmax

Absorbance of known nickel solution at Ni λmax

Absorbance of known Cobalt solution Co λmax

Absorbance of known Cobalt solution at Ni λmax

Calculations:

1. C Absorbance of unknown Mixture at Ni λmax

alc Absorbance of unknown Mixture at Co λmax
ulat
e the extinction coefficients for nickel at the nickel λmax (λ1) and for nickel at the cobalt λmax
(λ2).
2. Calculate the extinction coefficients for cobalt at the cobalt λmax (λ2)) and for cobalt at the
nickel λmax(λ1).
3. Write two equations at the two λmaxs as follows
ATotalλ1 =εi λ1bcNi + εoλ1 bcCo
ATotalλ2 = εi λ2 bcNi + εoλ2 bcCo

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