EXPT.

1: ANALYSIS OF A DOUBLE-COMPONENT MIXTURE

BY UV-VIS SPECTROPHOTOMETRY

OBJECTIVE
To resolve a two component mixture by absorbance measurements at the wavelengths of
maximum absorbance

INTRODUCTION AND THEORY

Frequently, it is desirable to determine the concentrations of two or more substances in the
same solution. If the absorption maxima of the two substances do not overlap, that is, if both
substances do not absorb light at each other’s absorption maximum, then the
measurements can be done by simply reading the solution absorbance at both wavelength
maxima of the two substances, and determining the absorbance from separate Beer’s Law
plots.

Very seldom does it happen though that two absorption spectra are independent of each
other in this manner. Usually at the wavelength maximum of component A, component B
almost always has at least some light absorbing character. Thus, the absorbances read at
the two wavelengths are composed of contributions from both components, and the problem
of how to separate them arises.

The solution lies in the fact that absorbance is an additive property of solutions. In other
words, the total absorbance of a solution of several components is equal to the sum of the
individual absorbances that would be measured if each component were separate in its own
cuvette.
Mathematically: Aλ1 = a1λ1 bC1 + a2λ1bC2 + …
where Aλ1 is the absorbance measured at wavelengthλ1; b is the path length;
a1λ1 and a2λ1 are the molar absorptivities of the various components at
wavelength λ1; and C1 and C2 are the concentrations of these components.

In this experiment, a mixture of cobalt and nickel will be resolved in this manner. For such a
case, two equations of the type just illustrated may be written:

A λ1= a1λ1Co b[Cu] + a2λ1Ni b[Ni]

A λ2= a1λ2Co b[Cu] + a2λ2Ni b[Ni]

The absorbances Aλ1 and Aλ2 are measured at the wavelength of maximum absorption for
both cobalt and nickel. In addition, the molar absorptivity values of both components at both
wavelengths are calculated by measuring the absorbance of pure solutions of the separate
components. With these data the above two equations become two equations in two
unknowns, C1 and C2, which may then be solved.

REAGENTS AND MATERIALS

0.06 M cupric nitrate , 0.06 M nickel nitrate
volumetric flasks (25, 50, 250 mL), pipets (10 mL), beakers , cuvette (quartz)

APPARATUS
Scanning double beam UV-VIS spectrometer

PROCEDURE

A. Spectral-Transmittance Curve
Prepare 250 mL of 0.06 M copper nitrate solution. Weigh the exact amount of the sample
needed and put it in a 100 mL beaker, then add about 20 mL of water to dissolve it. Stir
thoroughly until no solid particles are present. Transfer the solution into the volumetric flask
and then dilute to the mark with water. This is your stock solution.
1. Do the same to prepare the 0.06 M nickel nitrate solution.
2. Determine the wavelength of maximum absorbance for both Cu and Ni by scanning the
stock solution in the 200-1000 nm wavelength range.

B. Beer’s Law Check

1. Prepare 25-mL standard solutions of Cu and Ni with the following concentrations: 0.05 M,
0.04 M, 0.03 M, 0.02 M, 0.01 M and 0.005 M. Take the absorbance readings of each
solution at both wavelengths of maximum absorbance for the two metals. Check if Beer’s
Law is followed at the
twoλmax by plotting the absorbances of the solutions vs concentration. Determine the molar
absorptivities of Cu and Ni at both wavelengths.

C. Analysis of the Sample

Obtain a solution containing unknown amounts of Cu and Ni from your instructor. Read its
absorbance at the wavelengths of maximum absorbance. Compute for the amount in grams
of Cu and Ni in the aliquot. If needed, dilute the unknown sample to lessen the
concentration. Get a 10 mL aliquot and dilute up to 50-mL with distilled water. Measure the
absorbance at the wavelengths of maximum absorbance.
EXPT. 2: DETERMINATION OF MANGANESE IN STEEL SAMPLE USING UV-VIS
SPECTROPHOTOMETRY

OBJECTIVE
To determine the presence of manganese in steel sample using ultraviolet - visible
spectrophotometry

INTRODUCTION AND THEORY

Manganese in steel can be determined spectrophotometrically after oxidation to the purple

permanganate ion. The steel sample is dissolved in nitric acid and the oxidation is effected
with potassium periodate:

2 Mn2+ + 5 IO4- + 3 H2O → 2 MnO4- + 5 IO3- + 6 H+

Since the yellow iron (III) is present, phosphoric acid is added to form the colorless iron-
phosphate complex. If the steel sample contains cobalt or nickel, the colors of these interfere
in the manganese determination. The interference can be canceled by the addition of about
the same amounts of these elements to the standards as are in the unknown. Alternatively,
a sample can be carried through the entire procedure except for the periodate oxidation and
then used as a blank in setting the spectrophotometer to read zero absorbance.

The experiment first calls for determining a spectral-transmittance curve to find the
wavelength at which to perform the analysis. A check of Beer’s law is then made by
measuring the absorbances of permanganate solutions at several concentrations. The
unknown is determined by comparison with
the Beer’s law plot.

REAGENTS AND MATERIALS

Steel sample, conc nitric acid, ammonium persulfate, sodium sulfite, sulfurous acid, 85%
phosphoric acid, potassium periodate

Volumetric flasks (25, 100, 250 mL), pipet (5 mL, 10 mL), aspirator, erlenmeyer flasks,
beakers, hot plate, stirring rod

APPARATUS / EQUIPMENT
Scanning UV – VIS double beam ( Hitachi U-2000 )

PROCEDURE

(Note 1: Permanganate ion can undergo photodecomposition especially upon standing over
a period of time. Budgeting your time is of importance in this experiment. If time is not
adequate, avoid adding potassium periodate so as not to convert the manganese to
permanganate, or else, store your
solutions containing permanganate ion properly by wrapping the containers with foil.)

A. Spectral-Transmittance Curve

1. Secure a 250-mL volumetric flask. Weigh accurately (to the nearest 0.0001 g) 0.5g of
MnSO4·H2O (MM=168.94 g/mol) in a beaker. Dissolve in small portions of distilled water
and transfer the solution to the 250-mL volumetric flask. Rinse the beaker with portions of
water then add the rinsings to the volumetric flask. Dilute to the mark and shake thoroughly.
This will serve as the stock solution for the whole class. Label this flask appropriately as
calculated from the actual mass of MnSO4⋅H2O used (approximately 0.01184 M).
2. Place 10.0 mL of the stock solution, 25 mL of 85% phosphoric acid, 0.3 − 0.5 g of
potassium periodate, KIO4, in a beaker and dilute the solution with distilled water to about
100 mL. Boil the solution gently for about 3 minutes to effect oxidation to permanganate.
Cool the solution.
Transfer carefully into a 250 mL volumetric flask. Rinse the beaker three (3) times with 20
mL portions of water, adding the rinsings to the flask and dilute up to the mark. Check the pH
of the solution. If the pH value is greater than or equal to 7, add 6M nitric acid dropwise until
pH the is equal to or lower than 6. This solution will be used for wavelength scanning in step
A.4 and for the preparation of standard solutions in Part B.

3. Prepare a blank in exactly the same manner as in A.2 while omitting the addition of
potassium periodate. This blank will be used during wavelength scanning and when
measuring the absorbances of the standard solutions.

4. Measure the absorbance of the resulting solution (in A.2) from 440 to 700 nm, taking
readings at 20-nm intervals. In the region of maximum absorbance, take readings at 5-nm
intervals. Plot the absorbance vs. wavelength and connect the points to form a smooth
curve. Select the proper wavelength to use for the determination of manganese.

B. Beer’s Law Check (Standard Solution)

1. Prepare standard solutions with the following concentrations: 1.0 x 10 - 4 , M, 2.0 x 10

– 4
M, 3.0 x 10 – 4 M, 4.0 x 10 - 4 M; from the solution prepared in A.2. Measure the
absorbances of the standard solutions against the prepared blank.

2. Plot the absorbance vs. concentration for the four standard solutions. Draw the best
straight line between the points (use the least- squares method).

C. Analysis of Sample

(Note 2: Acid digestion must be done in the hood. Avoid inhaling the fumes generated while
digesting the samples.)

1. Weigh accurately (to the nearest 0.0001 g.) two portions of a 0.5-gram sample of steel
whose manganese content is to be determined. Dissolve each sample in 50 mL of 1:3 nitric
acid by heating and finally boiling gently for 1 to 2 min to remove oxides of nitrogen. Then
remove the flask from the burner/hotplate, add about 1 g of ammonium persulfate, and boil
the solution gently for 10-15 min.

(Note 3: If a precipitate of an oxide of manganese forms or if the permanganate color

develops, add a few drops of sodium sulfite or sulfurous acid and boil the solution a few
minutes to expel sulfur dioxide. The ammonium persulfate is added to oxidize carbon or
carbon compounds.
The excess persulfate is destroyed by boiling the solution. This reduces manganese to the
bivalent state. The solution should be clear).

Add about 10 ml of 85% phosphoric acid to each sample. To one sample, add 0.5 g of
potassium periodate. Dilute both solutions with distilled water to about 100 ml. [The first
solution is the sample for analysis and the second, which contains no periodate, is the
blank.] Boil the solutions gently for about 3 min to effect oxidation to permanganate. Then
cool the solutions and dilute up to 250 ml in a volumetric flask.

2. Measure the absorbance of the sample solution against the blank (prepared in C.1;
contains no potassium periodate) at the same wavelength used in second part to check
Beer’s law.
*Using the equation of the line, determine the concentration of permanganate and calculate
the percentage of manganese in the sample.
EXPT. 3: COMPLEX ION COMPOSITION BY THE CONTINUOUS VARIATIONS METHOD

OBJECTIVE

To determine the composition of Ni2+-ethylenediamine ( en ) complexes by method of

continuous variations.

INTRODUCTION AND THEORY

The solution composition of Ni2+-ethylenediamine (en) complexes can be determined

by the method of continuous variations. Generally, it is concerned with evaluating n for the
equilibrium,

Z + nL ↔ ZLn (1)

Both Ni2+ and the product Ni(en)n2+ complexes have absorptions in the visible region
of the emr spectrum, but their spectra are different.

Experimentally, the intensity of absorption at a given wavelength of a series of

solutions containing varying amounts of Ni2+ and en is measured. This absorbance is related
to the concentration of Ni(en)n2+ in solution. These solutions are prepared with the restriction
that the sum of the concentrations of Ni 2+ and en be the same for all solutions. In the case
where the equilibrium constant for the complex formation is very large, it is clear that the
intensity of the Ni(en)n2+ absorption will be greatest when the en concentration in solution is
exactly n times greater than that of Ni 2+. This is also true when the equilibrium does not lie
far to the right. Sufficient concentrations of the complex must, however, be produced so that
accurate absorbance measurements may be obtained on the solutions. It is therefore
possible to determine n and the composition of Ni(en)n2+ by knowing the ratio of en to Ni 2+ in
the solution that contains a maximum absorbance for Ni(en)n2+.

For the Ni2+-en system, a series of complexes are possible. The purpose of this
experiment is to determine which of these species are actually present in solution. The
possible equilibria involved follow:

Ni 2+ 1→ Ni(en ) 2+
K
+ en ←

Ni(en ) 2+ + en ←
2 → Ni(en ) 2+
K
2

Ni(en ) 2 2+ + en ← 3→ Ni(en ) 3 2+

K

Ni(en ) 3 2+ → Ni(en ) 4 2+ , etc

K4
+ en ← 

The values of the equilibrium constants will determine which species will predominate
in solution. Thus, if K2 is so much larger than K1 that virtually no Ni(en)2+ is present in
solution, the continuous variations method will identify Ni(en) 22+ without giving any evidence
of Ni(en)2+. Likewise, the relative size of K 3 with respect to K1 and K2 will determine what
species are characterized. One of the limitations of this method is the requirement that only
one equilibrium of the type in equation (1) be present in a solution of Z and L. Non-integral
values of n is obtained if more than one complex equilibrium is present in solution. For the
Ni2+-en reactions, this means that only two complexes [Ni2+ and Ni(en)2+, or Ni(en)2+ and
Ni(en)22+, and so forth can be present in any given solution. This will be true if K 1, K2, and K3
are greatly different. From potentiometric studies of the interactions of Ni 2+ and en, the
values of K1, K2, and K3 have been evaluated and they are in fact separated by large factors.
Although the method of continuous variations allows the determination of complex
compositions in this system, misleading or erroneous results might be obtained in a system
where the K values for successive equilibria are unknown.

If the absorbance at a given wavelength of each solution is plotted vs. the mole
fraction, X, of L in the solution, the maximum absorbance will occur at a mole fraction which
corresponds to the composition of ZLn. Hence, n is determined.

Assuming that substances Z and L react according to equation (1), equimolar solutions of Z
and L, each of M mol/L concentration, are mixed in varying amounts so that the total
concentration (Z and L) is M. A series of these solutions may be prepared by the addition of
X liters of L to (1-X) liters of Z (where X<1). The concentrations of Z, L and ZL n at
equilibrium in these solutions are designated C1, C2, and C3, respectively. Thus, for any
solution the concentrations are expressed as follows:

C1 = M(1-X) - C3 (2)

C2 = MX - nC3 (3)

C3 = KC1C2n (4)

where K is the equilibrium constant for reaction (1). The condition for a maximum in the
curve of C3 plotted vs. X is that

dC 3
=0 (5)
dX

Differentiation of equations (4), (3), and (2) with respect to X and combination of the three
resulting differential equations with equations (2), (3), and (5) gives

X
n= (6)
1− X

Therefore, from the value of X for which C 3 is a maximum, n may be calculated from
equation (6).

Now it remains to be shown that a maximum in the absorbance at a given wavelength of

light when X is varied coincides with the maximum of C3. From Beer-Lambert Law,

A = εbC (7)

The extinction coefficients of Z, L and ZL n at a given wavelength are designated ε1, ε2, and
ε3, respectively. Since the absorbance of a solution is the sum of the absorbances at that
wavelength of the contained species, the measured absorbance, Ameas, is

Ameas = (ε1C1 + ε2C2 + ε3C3) b (8)

If there is no interaction between Z and L, i.e., C3 = 0, the absorbance, AZ+L, would be

AZ+L = [ε1M(1-X) + ε2MX] b (9)

where M is still the molar concentration of the Z and L solutions.

The difference between Ameas and AZ+L is designated Y.

Y = [ε1C1 + ε2C2 + ε3C3 - ε1M(1-X) - ε2MX] b (10)

By differentiation of equation (10) with respect to X it can be shown that Y is a maximum

when C3 is a maximum if ε3>ε1, or a minimum when C3 is a maximum if ε3<ε1.

In this experiment Ni2+ corresponds to Z and en to L. Since en has no absorbtions in

the region of the spectrum under study, ε2 will always be zero. The cell to be used in the
measurements has a thickness of 1 cm. Under this conditions equation (10) reduces to

Y = Ameas - (1-X) AZ (11)

where AZ is the absorbance of the pure M molar Ni2+ solution. To evaluate n in Ni(en)n2+, a
plot of Y (ordinate) versus X (abscissa) at a given wavelength is made. A maximum in this
plot occurs at a certain X mole fraction. From this value of X, n may be calculated from
equation (6). Because more than one complex composition will be examined in this
experiment, plots will be constructed from data taken at several wavelengths corresponding
to different Ni(en)n2+ complexes.

REAGENTS AND MATERIALS

0.3 M nickel sulfate hexahydrate and 0.3 M ethylenediamine

Volumetric flasks (25, 500 mL), pipettes, cuvettes (quartz)

APPARATUS / EQUIPMENT

Scanning double beam UV-VIS spectrophotometer ( Hitachi U 2000 )

PROCEDURE

1. Prepare 500 mL of the following aqueous solutions: 0.30 M nickel sulfate

hexahydrate NiSO4⋅6H2O, 0.30 M ethylenediamine (en) C2H8N2. By mixing these
solutions, prepare mixtures having a total volume of 25 mL in which the mole
fraction of en, X, is 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9. Determine the
absorbances of each of these solutions and of the pure 0.30 M nickel sulfate
solution at the following wavelengths: 530, 545, 578, 622 and 640 nm. In using
the UV-VIS spectrophotometer ( Hitachi U 2000 ), the most convenient method of
determining the absorbances at the various wavelengths is to record the
spectrum of each solution from 500 to 650 nm. The spectra of all solutions can
be run on the same piece of chart paper.

2. Calculate Y values (from equation 11) at each wavelength for the entire series of
solutions, and make plots of Y versus X for each of the five wavelengths. From
the value of X at each of the five maxima, the values of n for the Ni(en)n2+
complexes may be calculated from equation (6).
EXPT. 4: POTENTIOMETRIC TITRATION OF PHOSPHORIC ACID

Objective

Solutions of pure phosphoric acid and of phosphoric acid mixed with its conjugate base,
dihydrogen phosphate, will be titrated with a strong base. The data from the titration of the
pure phosphoric acid will be used to determine the concentration of the acid. The titration of
the mixture will be done to illustrate the effect of the conjugate base on the titration curve.

Background

Acids that contain more than one acidic (ionizable) hydrogen (proton) are called polyprotic or
polybasic acids. The dissociation of polyprotic acids occurs in a stepwise fashion, one proton
lost at a time. For example, the generic triprotic acid will dissociate as shown in Reactions
(1) through (3). The equilibrium constants for these reactions are symbolized by K a. The
trailing subscript "a" indicates that the equilibrium constant describes an acid dissociation
reaction. The trailing subscript "n" is written as a number and indicates which proton is being
dissociated.

H3A + H20 <-----> H3O+ + H2A- Ka1 = [H3O+] [H2A-] / [H3A] (1)

H2A- + H20 <-----> H3O+ + HA2- Ka2 = [ H3O+] [HA2-]/ [H2A-] (2)

HA2- + H20 <-----> H3O+ + A3- Ka3 = [ H3O+] [A3-] / [HA2-] (3)

Reactions such as those shown in Reactions (4) and (5) are sometimes used as the basis of
chemical calculations, but these reactions are generally not considered to be representative
of what is actually occurring in solution. These reactions can be useful at times even though
they are not rigorously correct chemically. The equilibrium constant for Reaction (4) is the
product of Ka1 and Ka2 while the equilibrium constant for Reaction (5) is the product of K a1,
Ka2, and Ka3. Prove this to yourself.
H3A + 2H20 <-----> 2H3O+ + HA2- (4)

H3A + 3H20 <-----> 3H3O+ + A3- (5)

Remember that the numerical value of an equilibrium constant is an indication of how far to
the right a reaction proceeds. The greater the magnitude of the equilibrium constant, the
more complete the reaction. Generally the numerical value of K a1 is larger than that of Ka2,
which is larger than Ka3 This is reasonable when one considers that successive protons are
removed from species with progressively higher negative charges.

Polyprotic acids are called polybasic because they have more than one conjugate base. A
conjugate base is a species that is produced when an acid loses a proton. For example, the
species H2A-, HA2-, and A3- are all conjugate bases of H3A. The completely deprotonated
acid, A3-, is a weak base. The other two conjugate bases are also weak bases, but in
addition they have one or more acidic protons. As a result these species are amphiprotic or
amphoteric, which means that they can act as either an acid or a base. Molecules that are
partially deprotonated polyprotic acids are called acid salts. A common acid salt is sodium
bicarbonate, NaHCO3, which is found in baking soda.

When a strong base is added to a solution of a polyprotic acid, the protons of the acid are
neutralized in a stepwise fashion. That is, Reaction (6) will occur first until all of the H 3A is
used up. Then Reaction (7) will begin and will continue until all of the H 2A- is gone. Then
Reaction (8) will occur until all of the HA 2- is gone. This will only be true when the successive
dissociation constants are different by a large enough factor and when all of the acidic
species are strong enough. For example, phosphoric acid has K a values that are different by
a large enough factor to allow it to react with a strong base in a stepwise fashion, however,
the value of Ka3 for phosphoric acid is so small that the last proton of phosphoric acid is
extremely difficult to remove, and the reaction of phosphoric acid that is analogous to
Reaction (8) essentially will not occur in aqueous solution.

H3A + OH- <-----> H2A- + H20 (6)

H2A- + OH- <-----> HA2- + H20 (7)

HA2- + OH- <-----> A3- + H20 (8)

The titration curve for the titration of a polyprotic acid such as phosphoric acid will have the
form shown in Figure 1. This curve is a plot of pH (-log[H30+]) versus the volume of base
added. The first point on the curve corresponds to a solution of phosphoric acid only. The pH
at this point is due solely to the phosphoric acid in the solution. As soon as some base is
added, some H2PO4- is produced. The solution now contains both phosphoric acid (a weak
acid) and its conjugate base dihydrogen phosphate, and thus it is a buffer. A buffer is a
solution that is resistant to change in pH upon dilution and addition of acid and base. This
will be the situation from the initial point to the first equivalence point, and therefore this
region of the curve is called the first buffer zone. The pH of the point midway between the
first and second equivalence points is equal to the negative logarithm of K a1, pKa1. At the first
equivalence point, all of the H3PO4 has been neutralized and only H2P04- is present in the
solution. When more base is added, some of the H2P04- is neutralized and some HP042- is
produced. This solution contains both dihydrogen phosphate (a weak acid) and its conjugate
base hydrogen phosphate, and thus is a buffer. At all points between the first and second
equivalence points, the solution will contain these two species, and therefore this portion of
the curve is called the second buffer zone. The pH of the solution at the point halfway
between the second and third equivalence points is equal to pKa2. At the second equivalence
point the only species in the solution is HPO 42-. The region between the second and third
equivalence points is called the third buffer zone because the solution contains both the
weak acid HPO42- and its conjugate base PO43-. Halfway between the second and third
equivalence points the pH of the solution is equal to pK a3. At the third equivalence point, only
the weak base PO43- is in the solution. After the third equivalence point, the solution contains
phosphate ion and excess unreacted OH-.

Figure 1. Titration curve for titration of phosphoric acid with sodium hydroxide. The point
marked "a" is the initial point. The pH of the solution equals pK a1, pKa2, and pKa3at points b,
d, and f, respectively. Points c, e, and g are the first, second, and third equivalence points,
respectively. The points between a and c, between c and e, and between e and g are the
first, second, and third buffer zones, respectively.
Changes in the pH during the titrations you will do in this experiment will be monitored with a
glass pH electrode (probe). This electrode will generate a potential in proportion to the
hydronium ion concentration in the solution. The pH meter will electronically convert this
potential to pH. Titrations monitored with electrodes, such as glass electrodes, that measure
a potential are called potentiometric titrations.

In this experiment you will titrate a solution containing only phosphoric acid. The curve from
this titration will be used to calculate the concentration of the phosphoric acid and to
calculate the Ka1 and Ka2 values of phosphoric acid. You will also titrate a mixture of
phosphoric acid and potassium dihydrogen phosphate to discover the effect the added
species has on the titration curve of phosphoric acid.

Procedure:

1. Titration of H3PO4

Fill a 50.00 mL buret to the 0.00 mL level with standard 1 M NaOH. Then pipet 20 mL
of the phosphoric acid solution into a 100 mL beaker. Place the pH probe in the acid
letting the pH value equilibrate, then begin adding the NaOH in 1 mL increments for
each data time while carefully stirring with the probe. Continue adding NaOH until the
curve has peaked and leveled off. You do not need to record the final level off the
buret because you have passed the equivalence point by several milliliters.

2. Effect of KH2PO4 on the titration curve:

Add 20 mL of the phosphoric acid to a 100 L beaker and then mix in 20 mL of
distilled water. Titrate as before. Repeat with another 20 mL of phosphoric acid but
this time mix in 20 mL of 0.3 M KH2PO4 solution. Titrate as before.
EXPT. 5: DETERMINATION OF TRACE LEVELS OF COPPER AND IRON IN
VEGETABLE SAMPLES USING THE ATOMIC ABSORPTION
SPECTROPHOTOMETER

OBJECTIVE

To illustrate how trace amounts of several nutritionally important elements in vegetable

samples can be determined through atomic absorption spectro-photometry.

INTRODUCTION AND THEORY

Atomic Absorption Spectroscopy (AAS) is used for the qualitative and quantitative
identification and determination of trace levels of metals in different samples. In AAS,
measurement is made of the radiation absorbed by the nonexcited atoms in the vapor state.
It is similar to molecular absorption spectroscopy, the major difference being that unbound
atoms rather than molecules are the absorbing species. In terms of instrumentation, the
monochromator in an atomic absorption instrument is placed after the sample. This
arrangement is necessary to remove unwanted radiation created during the atomization
process.

In most common instruments, the sample solution is introduced into the flame in an
aerosol form. Before the salt vaporizes and dissociates into free gaseous atoms, the solvent
must first evaporate. At certain temperatures of an air-acetylene flame, atoms of many
elements exist mostly in the ground state. When a beam of radiant energy that consists of
the emission spectrum for the element that is to be determined is passed through the flame,
some of the ground state atoms absorb energy of characteristic wavelengths and are
elevated to a higher energy state. The amount of energy as a function of concentration of
an element in the flame is the basis of atomic absorption spectroscopy.

REAGENTS AND MATERIALS

copper metal or copper compound, ferric chloride hexahydrate FeCl 3.6 H2O, con.
nitric acid, samples: leaves of vegetables (Kangkong, Camote tops, Chili, etc.)

volumetric flasks (25, 50, 100, 250 mL), erlenmeyer flasks (250 mL), glass funnel,
beakers, pipettes, rubber aspirator, hollow cathode lamp (Cu and Fe)

APPARATUS / EQUIPMENT

Atomic Absorption Spectrometer (Varian SpectrAA 200)

PROCEDURE

A. Preparation of Stock Solutions

Weigh accurately to the nearest 0.001 g , 0.5 g of copper powder and 0.1 g ferric
chloride hexahydrate ( FeCl3 . 6H20), dissolve each in 20 ml of 1:1 nitric acid and dilute
up to the mark in separate 250 ml volumetric flasks. Then, get a 5 ml aliquot from both of
the prepared 250 ml solution and dilute separately to a 100 ml volumetric flask to obtain
100 µg/ml ( or ppm ) each of Cu and Fe stock solutions.

B. Preparation of Standard Solutions

Secure five (5) clean, 50 mL volumetric flasks and label with numbers 1 to 5. Place 0.50,
1.25, 2.50, 5.00 mL of the stock solution to flasks 1,2,3,4 respectively to prepare 1.00,
2.50, 5.00, 10.00 ppm of standard solutions. Dilute the solutions up to the mark with
distilled water. Flask 5 will serve as the blank.

C. Preparation of Sample

At least one (1) kilo of the vegetable sample is needed in this analysis (Note: Ask your
instructor for the kind of vegetable sample to be analyzed). Collect only the leaves from
the vegetable sample. Rinse the leaves and dry in the oven, maintain the temperature at
100 to 150 ºC for 30 to 45 minutes. Take the dried leaves out from the oven and weigh
accurately two grams of the dried sample. Place it in a 250 ml erlenmeyer flask and add
17.5 ml concentrated nitric acid. Prepare three samples. Boil slowly at low setting for 20
minutes and cool the solution. Add 10 ml distilled water and filter to a 50 ml volumetric
flask. Dilute the filtrate to the mark with distilled water passing through the filter paper.

D. Analysis of the Vegetable Sample

Record the absorbance of the standard solutions and the sample using the required

instrumental parameters (for Cu and Fe) of the Varian SpectrAA – 200. If the absorbance

is not within the range, get a 5 ml aliquot of the sample then, dilute to the mark in 25 ml

volumetric flask and take again its absorbance. Perform three trials.
E. Standard Addition Method

1. Secure five (5) clean, 50 mL volumetric flasks and label with numbers 1 to 5. To each
flask add 10 mL of the digested sample.
2. Place 0.00, 0.50, 1.25, 2.50, 5.00 mL of the stock copper solution to flasks 1,2,3,4 and 5
respectively to prepare 0.00, 1.00, 2.50, 5.00, 10.00 ppm of added standards. Dilute the
solutions up to the mark with distilled water. .
3. Record the absorbance of the each solution using the required instrumental parameters
(for Cu) of the Varian SpectrAA – 200.
4. Repeat procedures 1 – 3 but this time use iron as the standard to be added and set the
required parameters for Fe of the Varian SpectrAA – 200.
EXPT. 6: THE DETERMINATION OF SODIUM, POTASSIUM, AND CALCIUM IN
MINERAL WATER BY ATOMIC EMISSION SPECTROSCOPY

OBJECTIVE

To determine the sodium, potassium, and calcium content of a mineral water

sample by flame emission spectrometry.

INTRODUCTION AND THEORY

Flame emission spectrometry (FES) or flame photometry is particularly suited for the
determination of alkali and alkaline earth metals in aqueous samples or those that are
readily brought into solution.

If a solution containing a metal is aspirated in the form of an aerosol into a hot flame,
the solvent is evaporated from the droplets and the metal vaporized mainly as atoms. A
proportion of these atoms will be excited by the thermal energy of the flame and emit
electromagnetic radiation characteristic of the metal. The intensity of the emitted radiation,
measured at a suitable wavelength, is directly proportional to the concentration of the metal
in solution and hence in the original sample.

REAGENTS AND MATERIALS

calcium carbonate, potassium chloride, sodium chloride, magnesium chloride,

mineral water (different brands)

volumetric flasks (100, 1000 mL) beakers, pipette, stirring rod, aspirator, magnetic
stirrer, magnetic bar

APPARATUS / EQUIPMENT

Flame Emission Spectrometer ( Varian SpectrAA 200 )

PROCEDURE

A. Preparation of Solutions

1 Standard calcium solution, 500 ppm. Dry a quantity of CaCO 3 for about 1 hr at 110 oC.
Cool in dessicator; weigh (to the nearest milligram) 1.25g into a 600 ml beaker. Add
about 200ml of distilled water and about 10 mL of concentrated HCl; cover the beaker
with a watch glass during the addition of the acid to avoid loss due to spattering. After
reaction is complete, transfer the solution quantitatively to a 1-L volumetric flask, dilute to
the mark, and mix well.
2 Standard potassium solution, approximately 500 ppm. Dry a quantity of KCl for about 1
hr at 110°C. Cool; weigh (to the nearest milligram) about 0.95 g into a 1-L volumetric
flask, dilute to the mark and mix well.
3 Standard sodium solution, approximately 500 ppm. Proceed as in (2), using 1.25 g (to
the nearest milligram) of dried NaCl
4 Radiation buffer for the determination of calcium. Prepare about 100 mL of a solution
that has been saturated with NaCl, KCl, and MgCl2, in that order.
5 Radiation buffer for the determination of potassium. Prepare about 100 mL of a solution
that has been saturated with NaCl, CaCl2, and MgCl2, in that order.
6 Radiation buffer for the determination of sodium. Prepare about 100 mL of a solution that
has been saturated with CaCl2, KCl, and MgCl2, in that order.

B. Preparation of Calibration Curves

1. Add 5.00 mL of the appropriate radiation buffer to each of a series of 100 mL volumetric
flasks. Add volumes of standard that will produce solutions that range from 0 to 10 ppm
in the cation to be determined. Dilute to the mark with water, and mix well.
2. Measure the emission intensity for each solution. Aspirate distilled water between each
set of measurements. Correct the average values for background luminosity, and
prepare a working curve for the data.
3. Repeat for the other two cations.

C. Analysis of a Water Sample

Prepare duplicate aliquots of the unknown as directed for preparation of calibration
curves. If necessary, use a standard to calibrate the response of the instrument to the
working curve; then measure the emission intensity of the unknown. Correct the data for
background. Determine the cation concentration in the unknown by comparison with the
calibration curve.
EXPT. 7: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Determination of Caffeine in Beverages

OBJECTIVES

• To determine the concentration of caffeine in coffee, tea and cola beverage drink
using reversed phase HPLC.
• To gain experience in sample preparation and operation of a liquid chromatograph.

DISCUSSION

Food and pharmaceutical products are subjected to strict quality control (QC) procedures to
ensure consistency of the formulation within specified limits. Caffeine is a common
component of coffee and cola beverages.

High Performance Liquid Chromatography (HPLC) is used for the separation and
quantitative analysis of a wide variety of mixtures, especially, those where the components
are insufficiently volatile and/or thermally stable to be separated by gas chromatography
(CC). It is used extensively in the analysis of pharmaceutical products, foodstuffs and
beverages, agrochemicals, polymers and plastics and for monitoring drugs and their
metabolites in body fluids. The components of a mixture are carried through a column by a
mobile liquid phase pumped under high pressure. The order of elution is determined by the
chemical nature of components, the mobile phase and the stationary phase. Stationary
phases are silica or chemically modified silica (bonded phases) of a very small particle size
(3 mm to 10 mm). The eluted components are detected by monitoring the UV absorbance or
fluorescence, the current generated by redox reaction (amperometry) or the refractive index
of the mobile phase, eluted components are characterized by their retention times, tR , Or
their capacity factors, k' and quantitative analysis is accomplished by comparing the areas
of analyte peaks or heights with those of standards.

MATERIALS

HPLC with UV-Vis detector

Reversed phase column, C18

Volumetric flasks,100 mL and 25 mL

Syringe, 25 mL

Pipette, 1 mL and 10 mL

Rubber aspirator

Ultrasonicator

Analytical balance

Filter membrane , 0.45 mm

Caffeine standard, AR

Sample beverages

PROCEDURE
A. Caffeine standard

1. Into five clean and dry volumetric flasks (l00 mL), weigh accurately the following
quantities of caffeine : 2.5, 5.0, 7.5 and 10.0 mg.
2. Dilute to the mark with previously prepared methanol:water (20:80), adjusted to
approximately pi-I 3.50, with phosphoric acid. This is the same solvent to be used as
the mobile phase.

3. Shake the five caffeine solutions adequately to ensure dissolution and then degas
each for five minutes then filter using 0.45~1 filter membrane before injection into the
chromatograph.

4. Turn the pump and detector on. Set the pump flow rate at 2.3ml/min and the detector
sensitivity at 0.08 AUFS (absorbance unit full scale). Turn the recorder on and set at
slow speed rate. Prior to injection of the standards into the column, allow the mobile
phase to pass through the column for 5 to 10 minutes. Simultaneously record the
detector response to ensure that there are no substance left on the column from
previous experiments.

5. With syringe provided, inject 25 mL or more of caffeine standard starting with the
least concentrated. Take duplicate chromatograms for each of caffeine standard
solution.

B. Caffeine in tea and coffee

1. Into a clean, dry 25ml volumetric flask, pipette about 0.5ml coffee and into another
clean, dry 25ml volumetric flask , pipette 5 mi tea.
2. Dilute each volumetric flask to the mark with the methanol:water (20:80) solvent.

3. Follow steps 3 to 5 in procedure A.

C. Caffeine in cola beverage

1. Pour 10 to 15 mL of the cola beverage into a clean and dry beaker. Pour this into
another clean and dry beaker back and forth to original beaker until bubbling ceases.
Alteratively, the beaker can be place in an ultrasonicator for about 5 minutes until
bubbling ceases. The soda is now adequately decarbonated.
2. Into a clean and dry 25ml volumetric flask, pipette 10 mL of the cola beverage and
dilute to the mark with methanol:water (20:80) solvent.

3. Follow steps 3 to 5 in procedure A.

4. After the last chromatogram, flush the column with 50 mL of solvent (not at pH 3.50).

TREATMENT OF RESULTS

1. Measure the retention time, tR, Or volume VR , (tR and VR are directly proportional)
for
2. caffeine as the distance from the start of the solvent peak to the apex of the peak.

3. Measure the peak areas of the standard caffeine solutions by "triangulation" and
calculate the percentage composition of the mixture by "internal normalization", i.e.
cutting and weighing, height times width at half peak height or computing integrator.
Compare both sets of results with the original composition by weight.

4. Plot peak area vs. concentration for the standard solutions of caffeine and use it to
compute the caffeine contents of each sample .
EXPERIMENT 8: Gas Chromatography-Mass Spectrometric Analysis

Introduction
Gas chromatographic-Mass Spectrometric technique is an analytical tool that combined the
capability of chromatographic separation and characterization of components in a sample. It
can be used for both qualitative and quantitative analysis. It is a hyphenated analytical
technique that is used extensively in the medical, pharmacological, and forensic fields.

Procedure:

Weigh 500 g sampaguita flowers. Soak in AR hexanes for two days then filter. Repeat the
process twice. Concentrate the combined extracts in vacuo using a rotary evaporator.
Extract the concrete or concentrate with absolute ethanol, store in a freezer and filter.
Repeat ethanol extraction until no precipitation is observed. Concentrate the combined
ethanolic extracts in vacuo using rotary evaporator to give the absolute.

Analysis

Determine the λmas using UV-VIS

Determine the presence of probable compounds by Infrared analysis.

Identify volatile components of the sample using GC-MS by comparing the sample’s mass
spectra with the spectra library.

Compare all results and explain whether they reinforced each other or not.
EXPERIMENT 8: INFRARED SPECTROMETRY 1: SAMPLING METHODS AND
QUALITATIVE ANALYSIS

OBJECTIVE

To obtain and study the infrared spectra of a selection of compounds with a range of
common functional groups.

INTRODUCTION AND THEORY

Qualitative analysis using infrared spectroscopy is possible because of the unique

complexity of the infrared spectra of different compounds. The vibrational modes of motion in
a molecule give rise to the bands in the spectrum, and no two compounds give exactly the
same spectrum. There are, however, similarities between the spectra of similar compounds
or compounds with similar functional groups.

The frequencies associated with certain functional groups and certain substitution
patterns have been studied extensively. As a result, “correlation charts” have been
developed. These correlation charts give frequency ranges over which we can expect to find
vibrational bands for the molecular subgroups of interest; though the frequency ranges are
not all-inclusive. The vibrational frequencies of a molecule depend on the number, weight
and geometrical arrangement of the atoms and the force constant of each interatomic bond.
A change in any one of these factors will alter the infrared spectrum of a molecule.

The acquisition of a high quality spectrum is possible by the proper choice of sample
handling technique. It is also important to remember that the spectrum should have no
peaks which are ‘bottomed up’, that is, regions where the transmittance is near zero.

SAMPLING TECHNIQUES

Techniques for mounting the sample in the beam of the infrared spectrometer
depends on the physical state of the sample. Intermolecular forces vary considerably in
passing from solid to liquid to gas, and the infrared spectrum will normally display the effect
of these difference in the form of frequency shifts or additional bands, etc. It is, therefore,
most important to record on a spectrum the sampling technique used.

SOLIDS

There are three common techniques for recording solid spectra: KBr discs, mulls and
deposited films. Solids can be examined in solution but the solution spectra may have
different appearances from solid spectra since intermolecular forces will be altered.

Mull Technique

The sample is ground with the mulling agent in a mortar and pestle until a fine paste is
obtained. Liquid paraffin (Nujol) mull is the most widely utilized. The mull is then squeezed
between transparent plates/window. The thickness varies as the plates are rotated. To
disassemble a mull, one must not pull the windows apart but simply slide them. To prepare
a good mull, practice is necessary. Once perfected, this technique is the fastest and
easiest.

Pellet Technique

KBr discs are prepared by grinding the sample with dry KBr and compressing the whole into
a transparent wafer or disc. A few mg of the sample are mixed with 1 g KBr, a substance
which is highly transparent in the IR region. The mixture is subjected to pressure in a
hydraulic press. One disadvantage is the moisture pick up by KBr, giving rise to interfering
O-H frequencies.

Film Technique

Sample is cut into sheets of suitable thickness or melted and allowed to dry as a
film. This method is not often used due to the complexity of its preparation.

Solution: A solution in a volatile solvent is allowed to evaporate drop by drop on the

surface of the window. Polymers and various waxy or fatty materials often give excellent
spectra in this way.

LIQUIDS and SOLUTIONS

Thin Film Technique

The simplest technique of all consists of sampling a liquid as a ‘thin film’ squeezed between
two infrared – transparent windows ( e.g. NaCl or KBr ). The thickness of the film can be
adjusted by varying the pressure used to squeeze the flats together; the film thickness is »
0.1-0.3 mm. Care must be taken to keep the windows from moisture.

Solution Technique

Liquid samples can also be examined in solution. The sample can be dissolved in a
solvent and the spectrum of this solution recorded. The solution is placed in a ‘ solution or
liquid cell’ ( also known as cavity cell ) consisting of transparent windows with a spacer
between them of known thickness ; its thickness determines the path length of the cell –
usually 0.1 – 1.0 mm. A second cell containing pure solvent is placed in the reference beam
so that the solvent absorptions are cancelled out and the spectrum recorded is that of the
solute alone. The best solvent for use in IR measurements are nonpolar, nonprotic liquids
such as CS2 and CCl4. A more polar solvent such as CHCl3 may be useful except that the
C-H stretch regionmay be distorted.

Pure liquid samples or mixtures can also be injected in the liquid cell neat and high
quality spectrum obtained by choosing a suitable path length ( i.e. right spacer).

REAGENTS/MATERIALS

Acetone, benzophenone, unknown samples

APPARATUS/EQUIPMENT

Infrared spectrophotometer and accessories

PROCEDURE

Rules in Handling infrared plates/cells:

1. Do not touch the surfaces, they can be easily scratched. Handle by the edges only
and preferably wear gloves.
2. Do not breathe on the cell windows.

3. Do not remove the cells from the constant temperature room.

4. Avoid hygroscopic and moisture-rich substances.

Spectra Recording of Known Samples/Standards

1. Record the spectra of the samples provided using the different sampling techniques.
Identify and label the prominent bands in each spectrum.
2. Give the information about the chemical structure of a compound that can be
deduced from the IR spectra.

3. Compare the different sampling techniques. Comment on the kind of sample that can
be most appropriately prepared for each technique.

Spectra Recording of Unknown Samples

Obtain the infrared spectra of your unknown sample using at least two techniques
which you think are the most appropriate.

Data Processing

Interpret the infrared spectra obtained from the standards.

• Tabulate the results per technique per sample.

• Explain the bands observed for the standards.

• Discuss your observation on the different techniques for handling solid and liquid
samples. Compare the spectra obtained using each technique.

• Analyze unknowns one at a time.

• Tabulate the results using what you think is the most appropriate and best technique
depending on the sample.

• With the aid of the spectra of the standards and other known compounds, attempt to
deduce the principal structural or functional groups present.

• Show your reference table of frequencies in the final report.

EXPERIMENT 9: QUANTITATIVE ANALYSIS BY INFRARED SPECTROMETRY

OBJECTIVE

To determine the amount of acetone in isopropyl alcohol by quantitative infrared

spectrometry using the carbonyl band absorption.

INTRODUCTION AND THEORY

Although infrared spectrometry is primarily used for qualitative analysis, band

intensities are related to the concentration and path length of the sample through Beer-
Lambert law just as in the ultraviolet and visible regions of the spectrum. Because of the
complexity of infrared spectra and the effect of molecular interactions in solution on the
positions and intensities of some bands, the selection of suitable band upon which to base
quantitative measurements is often difficult. In addition, older instruments not having the
benefits of microprocessor control and ratio recording do not produce quantitative data of the
highest precision. However, two and even multi-component mixture analysis are feasible.
The determination of acetone, which is a common impurity in isopropyl alcohol as a result
facile oxidation, provides a good example:

( CH3 )2 CHOH  ( CH3 )2C=O + H2O

The strongest absorption band in the infrared spectrum of acetone is the C=O
stretching vibration at 1720 cm-1 which is in a region where isopropyl alcohol has very low
absorption. A series of acetone/chloroform standards can be used to prepare a Beer’s law
calibration plot based on this band and the concentration of acetone in a sample of isopropyl
alcohol determined after suitable dilution with chloroform.

REAGENTS AND MATERIALS

acetone, chloroform, isopropyl alcohol,

Volumetric flask (25 mL), pipette (1 mL), liquid or cavity cell, NaCl or KBr cell ( drilled and
undrilled ), glass syringe

APPARATUS/ EQUIPMENT

Fourier Transform Infrared Spectrometer ( Nicolet Magna 550 )

PROCEDURE

1. Prepare a series of five acetone standards in chloroform between 0.2% and 2.0%
 v.v., each in a total volume of 25 mL. Record the %T spectrum of each standard
between 1600 cm-1 and 900 cm-1 , flushing the cell thoroughly with chloroform
between each one and superimposing at least three scans on one chart. NOTE: 1)
Dry the volumetric flasks thoroughly before preparing your acetone standard
solutions; 2) Do not alter the 100 % control on the spectrometer during the
experiment and use the same cell throughout.
2. Prepare a 10 % solution of the sample of isopropyl alcohol in CHCl3 and record the
spectrum between 4000 cm-1 and 600 cm-1. If the absorbance of the carbonyl
stretching band is outside the range 0.3 to 0.7 absorbance, prepare another
solution diluted so as to bring the carbonyl absorbance with this range. Flush the cell
several times with CHCl3 and handle it with care.

3. For each standard and the unknown, draw a tangential baseline to the carbonyl
band and measure the difference in absorbance units between the peak maximum
and the point where a perpendicular drawn from the peak maximum intersects the
baseline (net absorbance )

4. Prepare a Beer’s Law plot from the net absorbance and concentrations of the
standards and determine the concentration of the unknown form the graph. Calculate
the molar absorptivity, Î , of the carbonyl band in m2mol-1 ( density of acetone =
0.1790 g cm-1 ).