CHEMISTRY PRACTICAL (CYI 102)

1st Year B. Tech (Common)

DEPARTMENT OF CHEMISTRY
IIT(ISM)
DHANBAD-826004
 SAFETY

Safety is our biggest concern in this course! You must read and know the section on Safety
before starting your first experiment. If you are ever unsure of a procedure, make sure you
ask the instructor or the TA.

General Information. A chemistry laboratory is a dangerous place. One must be aware of the
dangers and exercise extreme caution at all times. Before beginning work in the laboratory,
review the following rules. If you are in violation of any of the following rules, you will be
asked to leave the laboratory and will possibly be removed from the course. Eating, drinking,
and smoking are strictly forbidden in the laboratory.

Lab Attire. A lab coat is recommended. Protective gloves should be worn whenever the
potential exists for contact with toxic chemicals. Shorts and sandals are not safe lab attire,
since they provide no protection from splashed or spilled materials. Bare feet are absolutely
forbidden in the laboratory. To avoid entanglement with laboratory equipment, necklaces
and bracelets should not be worn, and long hair should be tied back.

Chemical Spills. Clean up chemical and water spills immediately. If the spill involves
dangerous chemicals, inform the instructor or the TA. Water on the floor can cause slips and
falls, and should therefore be cleaned up as soon as possible.

Check Glassware. Small cracks or "star-cracks" can cause glassware to break, explode, or
implode. Broken glassware should be turned over to the instructor or TA immediately.

Working with Chemicals. If you are unfamiliar with the properties, safe handling procedures,
or disposal requirements of any chemical, consult with your instructor or TA before you
attempt to use it. Dispose of all chemical waste in the appropriate containers that are
supplied.

Inappropriate Conduct. Disruptive behavior will lead to your immediate dismissal from the
laboratory.

Be Prepared. Be familiar with the task at hand

Keeping Your Laboratory Notebook

You should have a bound laboratory notebook, with numbered pages, is required for
this course (at least 60 pages). Your lab notebook is the primary record of all data and
observations generated during the experiment. It is regarded as proof of exactly what you
observed, and when the experiments were performed. All calculations (including, for
example, gross, tare, and final weights) should be recorded in your notebook - do not make
these on scratch paper. All entries should be in ink, and no erasing or white-out should be
employed. If an entry is to be disregarded, it should be deleted with a single line drawn
through it, such that it can still be read. Record any important observations that you think
would help someone else repeat your work. Procedures should describe what was done and
observed, not what you expected to do or observe. The notebooks will be graded in class.
Before you perform each experiment, your laboratory notebook should contain a pre-
lab write-up. This should include a title, a purpose, a list of reactants involved with molecular
weights, formulas, amounts in grams and moles to be used, and a list of balanced equations
including molecular structures that describe the reactions that you will be carrying out. After
the pre-lab section, the procedure section in your lab notebook should record what actually
happens. Here is where you make your observations about the details of the experiments,
including the entries of the weights of materials used and their physical appearance, and all
of the data recorded.

Grading
The grading will be based on lab note book/assignments/Lab reports related each
experiment*. The Lab Record Book has to be submitted not later than 14 days from the date
of the experiment.

Lab Reports
You should also have a hard bound Lab Record Book, in which the report of the experiment
carried out is written. Extreme care shall be taken while preparing this report. There should
not be any overwriting, cutting/erasing of the content. The report should be prepared in neat
hand-writing. The lab report shall be submitted approximately two weeks after the
experiment. The writing must be done only on the ruled page of the fair record.
Figures/graphs, if needed, shall be on the blank page. The Lab Report shall contain the
following components: Expt. No., Name of the experiment, Objective, Theory/Principle,
Procedure, Observations, Calculation, Result/Conclusion. ‘Expt. No’ shall be on top left corner
of the page. ‘Name of Expt.’ should be in bock letters, centralized and underlined. Each
‘subtitles’ should be in Block letters and underlined. Procedure shall be reported in passive
vice, simple past, past continuous or past participle depending upon the context. The
Observations/Readings shall be reported in Tables, the borders of which shall be prepared
using scale and pencil. If the results contain values, its units must always be reported. Each
student will submit one lab report.

Laboratory Knowledge
The knowledge and preparation of each individual student will be checked in class
throughout the course. Students are expected to be able to answer questions on the
techniques, procedures, motivations, and expected outcomes of the current experiment.

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Essential items
The students while entering lab should have a copy of the instructions sheet, Lab record, Lab
note book, A piece of cloth, lab coat, laboratory gloves & goggles etc.

*Includes preparation (pre-lab reading), knowledge of subject matter, cleanliness, conduct

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Experiments

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Experiment 01: Determination of total hardness of water by EDTA titration

Aim: To determine the Total Hardness of the given water sample by complexometric titration

Theory:
Water that contains dissolved hardness causing minerals, the most common ones are Calcium
and Magnesium, above 1 grains per gallon (gpg, which is equal to 17.1 parts per million
(ppm)). High levels of calcium and magnesium in water leads to several problems - more of
detergents are needed for cleansing, pipes and other appliances gets clogged, scale buildup
and mineral deposits in water heaters, undesirable taste in cooked foods, scum buildup in
clothes, skin and hair, etc. There are several methods to estimate the hardness of water. One
of the methods of estimation of Calcium and Magnesium is by using EDTA
(ethylenediaminetetraacetic acid).
The ethylenediaminetetraacetate (EDTA) is an example of a chelating agent. The later is a
polydentate ligand which can be coordinated to the metal ion at more than one point. The
EDTA molecule has up to six sites with which to coordinate to a metal ion/atom. For that
reason, EDTA forms stable complexes even with ions that typically do not form stable
complexes, such as calcium.

EDTA
Under proper conditions the complex with calcium ion is quite stable and the reactions of
calcium with EDTA proceed almost to completion.
Principle:
When Ca2+ is treated with the disodium salt of EDTA, Na2H2EDTA, a very stable complex is
formed.
Ca++ + H2EDTA2- CaEDTA2- + 2H+ (1)
Magnesium forms a similar complex, MgEDTA2-, which is far less stable than the Ca2+ complex.
When a sample containing calcium and magnesium ions is titrated with a solution of H2EDTA2-
the calcium ions are first complexed, forming CaEDTA2-. As more reagent is added and the
calcium ions are all combined in the complex, the magnesium ions form MgEDTA 2-. The

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desired endpoint of the titration is the point at which all of the Ca2+ and Mg2+ ions have been
complexed..
It has been found that the indicator Eriochrome Black T (H3In ) forms a colored complex with
Mg2+ and that this complex is less stable than MgEDTA2-. Consequently, reaction (2) occurs:
MgIn- + H2EDTA--  MgEDTA2- + HIn2- + H+ ………. (2)
Wine Red Blue
A color change is observed when the last of the indicator ions is displaced from its magnesium
complex. Since all calcium solutions do not necessarily contain magnesium ions, it is
customary to prepare the H2EDTA2- solution with a small amount of magnesium ion. When
added to a solution containing calcium ion and indicator, the magnesium complex first loses
EDTA to calcium (Eq. 3), and then the free Mg2+ reacts with the indicator (Eq. 4). After all the
calcium ion is complexed, reaction (2) occurs and a color change is observed.
MgEDTA2- + Ca2+  CaEDTA2- + Mg2+ ………(3)
Mg2+ + HIn2-  MgIn- + H+ ………(4)

Reagents required :
(i) M/100 EDTA solution,
(ii) Ammonia buffer (pH ~10) (Prepared by adding 6.75 g NH4Cl to 57 ml concentrated
ammonia and diluting to 100 ml ).
(iii) Erichrome Black-T indicator (mixed with solid NaCl)

Preparation of Reagents

(i) Preparation of M/100 EDTA solution :

 (EDTA is ethylenediamine tetraacetic acid. Di-sodium di-hydrogen ethylenediamine

tetra-acetate is available commercially of analytical reagent quality and can be used as
a primary standard.
 Its composition agrees exactly with the formula Na2H2C10H12O8N2.H2O (molecular
weight 372.25). It may be weighed out directly.
 Weigh accurately 3.7225/4 g (i.e. 0.9306 g) EDTA and transfer into 250 ml measuring
flask with the help of a funnel. Dissolve the salt in distilled water and make up the
volume with distilled water up to the mark.
 The total hardness of water is generally due to dissolved calcium and magnesium salts
and may be determined by the following procedure.

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Procedure:
 Take a clean burette, wash it first thoroughly with distilled water and then rinse it
thrice with the Standard M/100 EDTA solution taking about 5 ml portions of the
solution for each washing. Fill the burette now with the Standard M/100 EDTA solution
taking care that no air bubble is enclosed in the jet and the lower meniscus of the top
of the level of the liquid just touches the zero mark.
 Wash a 250 ml conical flask thoroughly with distilled water. Take 50 ml of the hard
water sample using pipette into conical flask, add 1 ml of the ammonia buffer solution
(pH~10) and a pinch of Eriochrome black T indicator until the solution become red.
 Now place the conical flask upon a piece of unglazed white paper just below the jet of
the burette, run in the standard M/100 EDTA solution slowly from the burette and
rotate the flask constantly until the colour changes from red to pure blue with no
reddish tinge remaining.
 Note down the burette reading. Repeat titration up to concordant reading.

Observations:

No. of Vol. of Hard Vol. of M/100 EDTA solution (in ml) Concordant
Expt. water (in ml) Reading
Burette reading
(in ml)
Initial Final Difference
1.
2.
3.

Calculation:

1 ml M/100 EDTA = 0.001 g of CaCO3

(Suppose 8.7 ml M/100 EDTA consumed for 50 ml hard water sample.)
Therefore, 8.7 ml M/100 EDTA = 8.7  0.001 g of CaCO3
Hence, 50 ml hard water sample = 8.7  0.001 g of CaCO3
106 ml hard water sample = (8.7  0.001x106) / 50 ppm

Result:
The Total hardness of the given water sample has been found to be …………….….. ppm.

Questions:
1. What are the main cause of water hardness?
2. Why EDTA used for the determination of hardness of water?
3. Which salt causes Hardness of water and why?

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Experiment 02: Verification of Beer-Lamberts law

Objective: To verify the Beer-Lambert’s Law and to determine the unknown concentration of
a given solution by colorimeter.

Theory:
 The Beer-Lambert’s Law states that when a beam of monochromatic light passes
through a solution, the decrease in the intensity of the transmitted light is directly
proportional to the concentration of the solution taken and the path length of the
solution.
 The logarithm of the ratio of the intensity of the incident light to that of the
transmitted light is called absorbance (A). It is proportional to the concentration (c) of
the solution and the path length (l). i.e.,
Log (Io/I) α cl
or, A= ε c l
 Where ε is the molar extinction coefficient which is a characteristic property a
chemical species absorbing the light. This law is valid only for dilute solutions.
 A plot of absorbance versus concentration will be a straight line passing through the
origin.

Equipment Required:

Colourimeter, cell, 100 ml volumetric flasks, Beaker and pipettes

Chemicals Required:

KMnO4 / K2Cr2O7, distilled water

Procedure:
 Prepare 50 ml 0.01 M KMnO4 or K2Cr2O7 solution (Stock Solution) by using distilled
water.
 Prepare 0.0001, 0.0002, 0.0004 and 0.0008 M different concentration solutions from
stock solution.
 By using the colorimeter measure the absorbance (A) of the prepared solutions.

Observation:

λmax of the solution = 440 nm for K2Cr2O7/540 nm for KMnO4

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Table

SL. No. Conc. (C) M Absorbance (a.u)

1 0.0001
2 0.0002
3 0.0004
4 0.0008
5 Unknown

Treatment of Data

1. Plot the graph of absorbance versus concentration which will be a straight line passing
through the origin.
2. Determine the concentration of the unknown solution from the plot by interpolating
its absorbance value with that of the concentration.

Result

The concentration of the given unknown solution is _____________ M.

Questions:
1. What is Lambert law and what is Beer law?
2. What do you meant by transmittance?
3. When Lambert-Beer law will not be obey?
4. What is the unit of ε?

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Experiment 03: Detection of functional groups in an organic compound for solid & liquid
sample

Aim:
To detect the functional groups present in the given set of organic compounds

Physical Property of the Sample:

1. State :
2. Color:
3. Odour:

Functional Group Analysis:

A. Test for –OH Group:
Experiment Observation Inferences

A drop of neutral FeCl3 Violet or Green -OH group is present.

solution is added to the coloration or precipitate
water or the alcoholic seen.
solution of the sample in
a test tube

Reactions:

B. Test for –CO2H Group:

Experiment Observation Inferences

i. The sample solution of i. The litmus turns i. –COOH group may

water in a test tube is red. be present.
touched with blue litmus
paper using a glass rod.
ii. A small portion of organic ii. –COOH group is
sample is sprinkled over present and
saturated aqueous confirmed.
solution of NaHCO3. ii. Effervescence of
CO2 comes out.

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Reactions:

C. Test for –NH2 Group:

Experiment Observation Inferences

100 mg of organic sample is dissolved in 5 ml of A brilliant red –NH2 group

dilute HCl in a test tube and cooled at 0-5 oC in an ice coloration present.
bath. Then pinch of solid NaNO2 is added to the observed.
mixture. The mixture is added to the ice cold alkaline
solution of beta-napthol.

Reactions:

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D. Test for >C=O Group:
Experiment Observation Inferences

Approx. 100 mg of organic sample is dissolve in A yellow or Carbonyl group

minimum quantity of ethanol in a test tube and 1 ml Orange (>C=O)
of Brady’s reagent (2,4-DNP: 2,4-Di nitro phenyl precipitation (aldehyde or
hydrazine) is added to it and shake vigorously. If no ppt observed ketone) present
happens then add a drop of conc. H2SO4 and scratching and confirmed.
the wall of the test tube with glass rod.

Reactions:

Conclusion: In the given organic sample (………………….) functional group(s) are present.

Questions:

a. How will you distinguish given compound is aldehyde or ketone?

b. What is Diazo-Coupling reaction?
c. What are test for carboxylic acidic groups?

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Experiment 04: POTENTIOMETRIC TITRATION

Aim: To determine the strength of the given acid by potentiometric titration

Theory:
When an electrode is dipped in an electrolyte solution, its potential depends on the
concentration of the ions to which the electrode is reversible. In an acid-base titration,
quinhydrone(QH) which is an equimolar mixture of quinone and hydroquinone is employed
as H+ reversible system, and the following cell is constructed:

Pt/acid + (QH) / / calomel

The reduction potential of quinhydrone and the EMF of the cell will depend on the pH of the
solution. On adding small aliquots of a base the EMF of the cell drops gradually; but, near the
quivalence point, again EMF changes in small decrements.

Apparatus Required:
potentiometer, platinum electrode, calomel electrode and connecting wires.

Chemicals Required:
stock solution of HCl, 0.1(N), oxalic acid, standardized NaOH solution (about 0.1 N),
quinhydrone.

Procedure:
1. Prepare 100 ml 0.1 N HCl: Take approximately 0.9 ml conc. HCl in a 100 ml volumetric.
Add 25 ml distilled water and mixed well to prepare homogeneous. Adjust final
volume of the solution 100 ml by adding distill water up to mark.
2. Prepare 250 ml 0.1 N NaOH: Take 1 g NaOH in a 250 ml volumetric flask. Add
approximately 50 ml distilled water and mixed well the solid to prepare homogeneous
solution. Adjust the final volume of the solution 250 ml by adding distill water upto
mark.
3. Prepare 100 ml 0.1 N oxalic acid: Take 1.2607 g NaOH in a 100 ml volumetric. Add 25
ml distilled water and mixed well to prepare homogeneous. Adjust final volume of the
solution 100 ml by adding distill water up to mark.
4. Standardized NaOH by oxalic acid solution: At first fill the burette by NaOH up to zero
marked. Check the burette properly; no solution must be leak out. Pipette out 10 ml
of 0.1 N oxalic acid in a conical flask. Add 2 drops phenolphthalein indicator. Note
down the initial point of the burette. Then, add NaOH solution dropwise from the
burette into the conical contained oxalic acid. Take the burette reading at which point
color is changed from colorless to pink color. Repeat titration up to concordant
reading. Then calculate the strength of NaOH by using following equation:
V1S1=V2S2
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 5. Pipette out 10 ml of the acid into a 20 ml clean beaker.
6. Add a pinch of Quinhydrone and mixed well by stirring.
7. Deep the electrodes into the solution.
8. Determine the EMF.
9. Run 1 ml aliquots of the standard NaOH from the burette and determine the EMF after
every addition.
10. At one point the EMF suddenly decreases. After that take few more standard NaOH
solutions.
11. The equivalence point lies in the range where the sudden decrease of EMF occurs.
12. Then repeat the experiment more carefully by adding 1.0 mL at a time in the range of
the equivalence point.

Treatment of Data:
1. Standardization of 0.1(N) NaOH solution:
Serial Volume of Volume Difference Concordant
no. 0.1(N) of NaOH Reading
Oxalic Acid Initial burette Final burette (mL)
(mL) reading (mL) reading (mL)
1.
2.
3.

 Strength of NaOH = …………..…N

 Volume of HCl = 20 mL.

2. Pilot run (1 mL addition):

Volume of NaOH EMF
(mL) (V)

3. Actual run: (0.1 mL addition):

Vol. of NaOH EMF (V) ΔE ΔV ΔE/ ΔV
(mL) (V) (mL) (V/mL)

Draw a graph of EMF Vs volume of NaOH added and dE/dV Vs. volume of NaOH added.

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Result:
(i) The volume of base required to neutralize the acid = ……mL
(ii) The strength of HCl = ……N.

Questions:
1. Name the reference electrode and write it′s electrode reaction?
2. What is role of salt bridge in a cell?
3. Write the Nernst equation for potentiometric redox reaction?

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Experiment 06: CONDUCTOMETRIC TITRATION

Aim: Determination of the strength of a solution of hydrochloric acid by a standard solution

of sodium hydroxide.

Theory:
Neutralization between a strong acid (HCl) and a strong base (NaOH) is represented by

H+ + Cl- + Na+ + OH- = Na+ + Cl- + (H2O)

Before NaOH is added, the conductance is high due to the presence of highly mobile hydrogen
ions. When the base is added, the conductance falls due to the replacement of hydrogen ions
by the added cation as H+ ions react with OHˉ ions to form undissociated water. This decrease
in the conductance continues till the equivalence point. At the equivalence point, the solution
contains only NaCl. After the equivalence point, the conductance increases due to the large
conductivity of OHˉ ions. If the conductance corresponds to the volume of NaOH solution
added be plotted, two straight lines having opposite slopes would be obtained. The point of
intersection of the two straight lines would give the equivalence point. The strength of NaOH
solution should be at least 5 times greater than that of the HCl solution, so that the
effect of volume change on the conductance be negligible.

Apparatus Required:
Conductometer

Chemicals Required:
stock solution of HCl, 0.1(N), oxalic acid, standardized NaOH solution (about 0.1 N),
quinhydrone.

PROCEDURE:
 Preparation of 100 ml (N\10) standard oxalic acid solution.
The equivalent weight of oxalic acid = 63. So, 1000 ml of 1(N) oxalic acid solution will
contain 63.0 gm oxalic acid. About 0.63 gm of oxalic acid is weighed from a weighing
bottle by difference and is poured into 100 ml volumetric flask. Dissolved it in small
volume of water by shaking and the volume is made up to the mark with distilled water
and thoroughly shaken to prepare 100 ml (N\10) standard oxalic acid solution.
 Standardization of supplied NaOH solution with a standard oxalic acid solution:
10ml of the standard oxalic acid solution is pipetted out into a 250ml conical flask and
about 1-2 drops of phenolphthalein indicator was added. The NaOH solution is titrated
against this solution with the endpoint being colorless to pink. Titration is done
thrice. Data is recorded in Table 3.
 Conductometric titration:
10ml of supplied HCl solution (strength in the order of N\10) is pipette out into a
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 250ml beaker and about 100ml of distilled water is added. The burette was
filled with NaOH solutions and number of drops of NaOH solutions corresponding
to a measured volume was calculated. From this calculation 1 drops corresponding
to how much volume is estimated. The conductivity cell is inserted into the acid
solution in the beaker in such a way that the two electrodes completely dipped into
the solution .The cell are connected to the digital conductometer to measure the
conductance of the solution. The initial conductance of HCl solution is noted and
then NaOH solution is added drop wise from the burette, 10 drops at a time in the
beginning, 4 drops at a time near the end point. Near the end point there is a sharp
rise in conductance. Beyond the end point 6 to 7 more reading by adding 10 drops
of NaOH solution at a time is taken. After each addition of NaOH solution the
beaker was shaken gently, waited for a minute and conductance was noted. The
conductance against corresponding no. of drops of NaOH solution added are given
in Table 4.

Result

1. Recording of temperature:
Initial temperature(0c) Final temperature(0c) Mean temperature(0c)

2. Strength of Oxalic Acid:

Weight taken (gm) Weight to be taken (gm) Strength (N)

0.63

3. Standardization of supplied NaOH solution with standard Oxalic Acid:

SI No: Oxalic Acid Burette reading for NaOH Mean value of
solution (ml) NaOH
taken(ml) Initial burette Final burette Volume of used(ml)
reading reading NaOH used
(ml) (ml) (ml)
1.

2.
3.

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4. CONDUCTOMETRIC TITRATION:
No. of No. of drops of NaOH
observation Observed Conductance solution added
(ms/cm)

( Add extra sheet if required)

 CALCULATION

 Strength on NaOH solution

 1ml of burette =………… drops.

 1drop of burette = ………….ml.

Graph:
Conductance against corresponding no. of drops of NaOH solution added are plotted in
graph. It shows the conductometric titrations curve of HCl solution by NaOH solution.

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Calculation from the Graph:

Conclusion:
From the conductometric titration the strength of the supplied HCl solution is found to be
…………………. (N) at ……………°C.

Questions:
1. What is cell constant? What it′s unit?
2. What is the change in conductivity when a solution is diluted?
3. Explain why conductivity increases after endpoint in acid-base conductometric
titration?

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Experiment 06: Acid-Base Titration

Objective: Determination of the concentration (strength) of a given sodium hydroxide solution

by titrating it against a standard solution of oxalic acid.

Principle:
In the titration of a strong acid with a strong base, the amount of acid and base becomes
chemically equivalent at the end point and the chemical reaction is called neutralization
reaction. Near the end point there is a sudden change in the pH of the solution. If after end
point even a small amount of base/acid is added the solution would become slightly alkaline
or acidic respectively. In the titration between oxalic acid (weak acid) and sodium hydroxide

(strong base), following reaction takes place:

In this titration phenolphthalein (HPh) is used as an indicator. The concentration of unknown

solution is calculated in g/L. Molarity of the solution can be calculated by using the formula:

a1M1V1 = a2M2V2

Where a1, M1, V1, are respectively basicity, molarity and volume of acid used and a2, M2 and
V2 are acidity, molarity and volume respectively of base used in the titration.

Procedure:
1. Prepare 0.1 M Standard Solution of Oxalic Acid.
2. Titration of Sodium Hydroxide and Oxalic Acid Solution
 Clean the burette thoroughly, wash it with distilled water and finally rinse it with
sodium hydroxide solution. (Always rinse the burette (Fig.) with the solution,
which is to be taken in it). Clamp the burette vertically in a burette stand.
 Fill sodium hydroxide solution into the burette through a funnel above the zero
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 mark.
 Remove the air gap, if any, from the nozzle of the burette by running the solution
forcefully from the burette nozzle.
 Remove the funnel before noting initial reading of the burette. Also while noting
the reading, see that no drop of the liquid is hanging at the nozzle of the burette.
 Note the initial reading by keeping the eye exactly at the same level as the
meniscus of the solution.
 Pipette out 10 mL of oxalic acid solution in a washed and dried conical flask. Always
wash the pipette with water and rinse with the liquid to be measured before
pipetting out the liquid.
 Add 1-2 drops of phenolphthalein indicator to the conical flask. Place the flask over
the glazed tile as shown in Fig. Titrate the acid with sodium hydroxide solution till
a very faint permanent pink colour is obtained. Add sodium hydroxide solution in
small amounts initially and then dropwise.
 Read the lower meniscus of the solution in the burette again and record it as final
reading.
 Repeat the procedure until three concordant readings are obtained. Record your
readings as in Table.

Table: Titration of sodium hydroxide vs oxalic acid solution

Sl. Volume of oxalic acid Burette readings Volume of sodium Concordant
No. solution taken in Initial Final hydroxide solution reading in
conical reading reading used mL
flask each time (V1 mL) (x) (y) V2 mL = (y–x) mL

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Calculations:

Result:

The concentration of NaOH solution is ——————— g/L.

Questions:
1. What is an indicator?
2. Define neutralization reaction?
3. Consider titration of 10 ml of 0.1 (M) HCl with 10 ml 0.1 (M) NaOH-
4. What salt is formed during this reaction? Do you expect salt solution at equivalence
to be acidic, neutral or basic?

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Experiment 07: Thin Layer Chromatography

Aim: Determination of the number of compounds present in the supplied sample by TLC.

Theory:
Mixtures of compounds are very common in Organic Chemistry. Most reactions produce more
than one product. Naturally occurring materials are only rarely 100% pure. It is therefore
desirable to have a simple, fast and efficient way to determine the purity of Organic mixtures.
The separation of a mixture by passing it, in solution, over an adsorbent (such as Alumina or
Silica Gel) is the basic idea of Chromatography.
Chromatography is a very general phenomenon. It involves the passage of a mobile
phase across a stationary phase in a column. Usually a mixture of compounds is present in the
mobile phase. As soon as the mixture comes in contact with the stationary phase, some or all
of the components of the mixture are adsorbed on it. As additional mobile phase comes along,
some or all of the mixture will dissolve and continue moving. This adsorption/solution process
continues along the length of the column. If a proper choice of mobile phase, stationary
phase, solvent and other operating parameters was made, the mixture will be separated in
the column and its various components will emerge at different times.
In Thin Layer Chromatography ("TLC"), a liquid solution is directly applied to a solid
adsorbent. Capillary action draws a developing solvent up the TLC plate. As this solvent passes
through the spot, the mixture will be dissolved and will begin to move with the solvent front.
However, the adsorbent will also reabsorb part or all of the mixture. As more solvent comes
by, the mixture will again go into solution, move further and be reabsorbed. Since different
materials will be dissolved and reabsorbed at different rates, separation will take place. The
slide is removed from the chamber once the solvent front reaches a predetermined spot near
the edge farthest from the point of spotting. This passage of the solvent front through the
adsorbent is known as developing the plate. The extent of separation, measured by retention
factor ("Rf ") value differences, will depend on the relative solubilities and relative strengths
of adsorption of the components of the mixture.
Organic compounds interact with absorbents by a variety of interactions. If the
compound is non-polar, it can only have weak 'Van der Waals' attractions for the absorbent.
However, more polar molecules may interact more strongly by a variety of mechanisms
including dipole-dipole interactions, coordination, and hydrogen bonding. The most
important rule of chromatography is that the more polar compounds will be absorbed most
strongly on absorbents (stationary phases), while non-polar compounds will be only very
weakly absorbed.
In a typical chromatography experiment, the non-polar compounds, since they are
poorly absorbed, will be held least strongly and will move quickly through the plate. Polar
compounds, on the other hand, will be slowed on their process through the plate by their
strong interactions with the solid phase. This separation based on polarity will explain most
of the chromatography encountered in this course.
23 | P a g e
Types of Adsorbents used in Chromatography
Listed in decreasing power of adsorption:
 Alumina,
 Activated Charcoal,
 Magnesium Silicate,
 Silica,
 Starch
Just as we have a variety of stationary phases to choose, we also have an even larger
assortment of mobile phases (or, eluting solvents). They are also categorized according to
their ability to move polar compounds through the chromatography column.
Solvents Commonly Used in Chromatography
Listed in decreasing polarity:

 Acetic Acid
 Water
 Methanol
 Acetone
 Ehtyl acetate
 Diethyl ether
 Chloroform
 Methylene chloride
 Toluene
 Cyclohexane
 Petroleum ether

For a typical separation, a variety of different combinations of solvent and adsorbent may
be effective. However, these combinations are only obtained by trial and error, based on
experience. There is no magic formula that will allow prediction of just the right set of
conditions for any given separation.
Once you have developed your plate, since most compounds are colorless, the location of
the separated samples, or spots, is usually not apparent. The plate must be visualized. This
visualization may be accomplished in a number of ways. If the compound(s) fluoresce, shining
a UV light on the plate may indicate the location of the separated spots. Conversely, the
adsorbent may be made to contain a small amount of a fluorescing substance. When the
developed plate is exposed to a UV lamp, most of the plate will fluoresce one color. Wherever
a spot is located there will be either a different color or less fluorescence. While the UV light
is ON, the position of the visualized spots is sketched on the plate with a sharp pencil.
Alternatively, visualization may be accomplished by reacting the developed plate with a
chemical reagent. Iodine (I2) is one of the easiest to use of the several common chemical
24 | P a g e
visualizing agents. The developed slide is simply exposed to I2 vapors in a chamber similar to
the developing chamber for a few minutes.
Almost all compounds will form a weak colored complex with the I2. This complex will
appear as a darker area on the slide. Again, the darkened areas are traced with a pencil before
the I2 evaporates and the color disappears.
This TLC technique usually requires only a few minutes for a complete analysis, and
requires only about 10 microliters of the solution to be analyzed (a microliter is a millionth of
a Liter (10–6 L, or 10–3 mL)). A few mL of the developing solvent is placed in a simple chamber,
such as a 4-oz wide mouth jar. To insure an atmosphere saturated with the developing solvent
in the chamber, a piece of filter paper is also present to act as a wick and the chamber is kept
capped except when adding or removing a TLC plate.
The 'spots' are characterized by their Rf value, a measure of how far the spot traveled with
that combination of adsorbent and solvent.
Rf values will change when either of these factors is changed.

Rf =

Distance spot traveled

Distance Solvent Traveled
On this scale, TLC is only an analytical tool, albeit a very valuable one. If samples of the
separated materials were desired, the entire experiment could be scaled up to allow
milligrams to be separated. The plates would be larger and the amount of adsorbent would
be increased, but the procedure would be the same. The spots would be visualized by UV
(non-destructive) and then separately scraped from the glass plate. The samples could be
recovered from the adsorbent by extracting the scrapings with a pure solvent such as ether,
and then carefully evaporating the solvent.

Aperatus required :
TLC plate, developing chamber, capillary tube.

Reagents required :
Hexane, acetone, iodine (solid), measuring cylinder

Procedure
 Obtain a TLC plate and a developing chamber.
 In the developing chamber, place about 5 mL of the TLC developing solvent (7:3
mixture of hexane : acetone). Be sure the depth of solvent is no more than 0.5 cm. If
the start line should touch the solvent directly, the TLC experiment is ruined since
some or all of the sample will be dissolved into the solvent pool.
 Stopper the chamber to allow it to become saturated with solvent vapors. A cap is
kept in place at all times, except when adding or removing a plate. Allow at least 5-10

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 minutes for the chamber to equilibrate before the first plate is developed.
 Trace a light pencil line about 1 cm from the bottom of the plate and another light
pencil line 5 cm up the plate from the first line. You should make your pencil marks on
the papery side (not on the side with the glossy finish).
 Use a microcapillary tube to load the sample onto the TLC plate. Allow the tip of the
drop at the end of the capillary to just touch the plate. Blow lightly onto the plate after
each drop is added to allow the solvent to evaporate. Each time you add sample to
the spot, make sure it never gets any larger than it did the first time. This will ensure
a very concentrated spot at the start line and will give the most concentrated spots
(nearly round) on development of the plate.
 After all spots have been applied, and all spots are dry, the plate may be placed into
the developing chamber and capped immediately to avoid loss of the solvent
saturated atmosphere. Almost immediately, the solvent will begin to migrate up the
plate.
 Once the solvent reaches the top line on the plate, remove it and allow the plate to
dry.
 As the plate dries, you will notice a change in its appearance. However, the spots will
not be visible unless they are colored materials. The spots must be visualized. A UV
lamp is the simplest way to visualize.
 Place the dry plate on the bench top and allow the UV light to shine on it. If there is a
spot, it will probably show as a different color of fluorescence than the background,
or as a darkened area on the adsorbent.
 With a very sharp pencil or other sharp instrument, draw an outline of each spot in
the adsorbent. Turn OFF the UV lamp and carefully put it away. Include your TLC plate
with your lab worksheet.

Calculation:
 Calculate the retention factors for each one of the pigments on your plate.

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Result: The Rf valud of the supplied samples is ……………

Questions:

1. What is Rf value?
2. Explain the term “stationary phase” and “mobile phase”?
3. You are given a sample that is believed to be either cis or trans-stilbene. Would TLC
be a good method to determine which compound you have? Why or why not?

27 | P a g e
Experiment 08: DETERMINATION OF VISCOSITY OF A LIQUID

Objective: To determine the viscosity of a given liquid using Ostwald Viscometer

Theory:
Liquid differ considerably in the case with which flow. This measure the resistance to flow of
liquids is determined by its viscosity. It deemed to be a frictional effect caused by molecular
attractive forces acting when one layer of liquid passes over another. The coefficient of
viscosity is defined as the force required per unit area to maintain an unit difference of
velocity between two parallel liquid layers, 1 cm apart. The unit of viscosity is poise and the
coefficient of viscosity is dynes per cm2. The viscosity of a liquid is usually measured by timing
it rate of flow through a capillary tube under a definite pressure employing and Ostwald’s
viscometer. The relationship is expressed as:

η1 / η2 = d1 × t1 / d2 × t2,
where d1, d2 and η1 and η2 are the density and viscosity of the liquid.

Procedure:
 The viscometer is cleaned by chromic acid followed by washing in water and rinsing in
acetone. The viscometer is clamped vertically and distilled water is introduced with a
pipette in such a way, that the lower reservoir is nearly half- full. Using the rubber
tube, raise the water by sucking out the water into the enlarged upper bulb, until it
reaches watermark.
 Allow the water flow down the capillary. Start a stopwatch as soon as the descending
water level touches the upper marks and stop it when the level touches the lower
mark. Record the time taken. Repeat the procedure thrice. The viscometer is cleaned
and dried and the same procedure is repeated using the given liquid.
Observation:
Viscosity of water =
Viscosity of Experimental Liquid =
Density of water =
Density of Experimental Liquid =
Temperature =

Sl. No. Liquids Time Density Viscosity

(in seconds) (g/ml) (poise)
1. Water
2. Experimental Liquid

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Calculation:

Time flow of water = t1; Density of water = d1; Viscosity of water = η1

Time flow of Exptl. Liquid = t2; Density of Exptl. Liquid = d2;
Viscosity of Exptl. Liquid = η2
By applying the formula η1 / η2 = d1 × t1 / d2 × t2,
We can calculate the value of η2.

Result: The viscosity of the exptl. liquid (η2 ) is ………………………..Poise at ……………°C.

Questions:

1. Define viscosity?
2. What is the viscosity coefficient? What it′s unit?
3. How does the viscosity of the fluid changes with temperature?

29 | P a g e
Experiment 09: DICHROMATIC TITRATION
Object: To determine the strength of salt by dichromatic titration.

Theory:
Ferrous ammonium sulfate [FeSO₄, (NH₄)₂SO₄, 6H₂O] is a stable Mohr‫ﹸ‬
s salt. Acidic potassium
dichromate solution is a strong oxidizing agent and rapidly reduced by ferrous ion at room
temperature to a green chromic salt when added to ferrous ammonium sulfate solution
containing dilute sulphuric acid. In this reaction ferrous sulfate is oxidized to ferric sulfate,
ammonium sulfate remains unreacted.
K₂Cr₂O₇ + 4H₂SO₄ → K₂SO₄ + Cr₂(SO₄)₃ + 4H₂O + 3[O]
6FeSO₄ + 3H₂SO₄ + 3[O] → 3Fe₂(SO₄)₃ + 3H₂O
K₂Cr₂O₇ + 6FeSO₄ +7H₂SO₄ = 3Fe₂(SO₄)₃ + K₂SO₄ + Cr₂(SO₄)₃ +7H₂O

N-phenylanthranilic acid is used as an indicator when all Fe⁺² ions have been converted to
Fe⁺³ ions, the color change of the solution changes from greenish to purple.

Apparatus Required:
250 ml measuring flask, burette, pipette, 100 ml conical flask.

Chemical Required:
Ferrous ammonium sulphate solution, 0.1 N standard potassium dichromate solution, 2 N
H₂SO₄, N-phenylanthranilic acid.

Procedure:
 Prepare 250 ml 0.1 N standard potassium dichromate solution: Take 1.2 g of K₂Cr₂O₇
in 250 ml measuring flask. The solid is wash down by adding distilled water. The flask
is shaken until K₂Cr₂O₇ dissolve. Adjust final volume of the solution 250 ml by adding
distilled water upto mark.
 Rinsed burette and filled up with potassium dichromate solution upto initial mark.
 Pipette out 10 ml of ferrous ammonium sulphate solution and taken into a 100 ml
conical flask.

30 | P a g e
  10 ml of 2 N H₂SO₄ solution added in conical flask (step-3).
 Add 2-3 drops of N-phenylanthranilic acid into solution of conical flask.
 Titrate the solution mixture with constant stirring against dichromate solution until
the colour of solution changes from greenish to purple by a single drop addition.
 Titration repeat for several time.

Treatment of data:
Titration of ferrous ammonium sulfate against potassium dichromate solution:
Sl. No. Vol. of salt Burette reading (ml) Vol. of Burette reading
solution taken Potassium
V2 (ml)
V1 (ml) Initial Final dichromate
solution require
(ml)
1.
2.
3.

Calculation:
Let us strength of ferrous ammonium sulphate solution =N₁
The strength of potassium dichromate solution (N₂) = 0.1 N
We know N₁V₁ = N₂V₂
Where, V₁ = Volume of ferrous ammonium sulphate solution = 10 ml (taken)
V₂ = Volume of potassium dichromate solution
𝑁2 𝑉2
Therefore, 𝑁1 = 𝑁
𝑉1

The strength in g/L = N₁ × 392.1 (Equivalent weight of ferrous ammonium sulphate)

Result:
The strength of ferrous ammonium sulfate solution is……………….g/L.

Questions:
1. What is dichromatic titration?
2. What is the difference between endpoint and equivalence point?
3. Which indicator can be used instead of N-phenylanthranilic acid?
31 | P a g e
Experiment 10: SYNTHESIS OF MOHR’S SALT

Aim: To synthesis of Mohr’s Salt

Theory:
The reaction of ferrous sulfate and ammonium sulfate to form ferrous ammonium sulfate
(Mohr‫ﹸ‬s salt). The formation of Mohr‫ﹸ‬
s salt shown as follows:

FeSO₄ + (NH₄)₂SO₄ + 6H₂O → FeSO₄·( NH₄)₂SO₄·6H₂O

Ferrous ammonium sulfate (Mohr′s salt)

Fe⁺² ions undergo hydrolysis while preparing an aqueous solution of ferrous sulfate in water
dilute sulphuric acid is added to prevent hydrolysis of the salt.

Apparatus Required: Beaker, one 50 ml conical flask, glass rod, tripod stand, funnel, filter
paper.

Chemical Required: Ferrous sulfate, ammonium sulfate, dilute sulphuric acid, ethanol, water.

Procedure:
1. Take 3.5 g ferrous sulfate and 1.7 g of ammonium sulfate in 50 ml conical flask
containing 5 ml of distilled water. Heat it until it gets dissolve. Add 0.5 ml of dilute
sulphuric acid to the flask and concentrate the solution by heating till the
crystallization point is reached.
2. Allow the mixture to cool at room temperature.
3. On cooling, light green crystals of ferrous ammonium sulfate separate out.
4. Decant the mother liquor and wash the crystals by shaking with a very small amount
of 1:1 water and alcohol mixture to remove sticking mother liquor.
5. Separate the crystal by filtration using a funnel having filter paper and wash with
alcohol. Dry them between the folds of filter paper and note down the yield.

Result: Yield of the Mohr‫ﹸ‬

s salt is .............%/g.

Questions:
1. What is double salt? Give two examples?
2. Why is water used in the preparation of Mohr′s salt?
3. Why dilute sulphuric acid added to the solution during the preparation of Mohr′s
salt?

32 | P a g e
Experiment 11: SYNTHESIS OF ASPIRIN

Aim: To synthesis of the aspirin

Theory
Salicylic acid undergoes acetylation with acetic anhydride in the presence of an acid catalyst
which generates acetyl carbocation. The acetylation takes place at the weakly acidic group in
preference to the strong carboxylic acid group to form aspirin.

Apparatus Required:
One 500 ml beakers, two 250 ml beaker, 250 ml round bottom flask, funnel, glass rod,
watch glass, filter paper.

Chemical Required:
Salicylic acid, acetic anhydride, conc. Sulphuric acid, ethanol, distilled water, ice.

Procedure:
1. Take 3 g of salicylic acid in a round bottom flask.
2. Take 5 ml of acetic anhydride and few drops of conc. Sulphuric acid in a beaker. Add
this mixture into round bottom flask containing salicylic acid (step-1).
3. Heat this mixture for 20 minute on water bath.
4. This hot mixture is added to the ice cold water and stir vigorously.
5. After cooling, filter the mixture by using a funnel with filter paper to obtain crude
aspirin.
6. For recrystallization, take crude aspirin into a beaker. Add 10 ml ethanol and 25 ml of
water. Warm the mixture until aspirin get dissolve after that remove the heat and cool
the beaker.
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 7. Again filtration occur using funnel containing filter paper and wash out with water.
8. At the end remove filter paper and allow it to dry on a clean watch glass.
9. Determine the mass of crystal.

Result: Yield of aspirin is……………….%/g.

Questions:

1. Why did acid use as a better catalyst?

2. What unwanted reaction would happen if the reaction mixture was heated too long
when trying to get rid of the organic reaction?
3. What is the consequence of recrystallization?

34 | P a g e
Experiment 12: KINETICS OF ESTER HYDROLYSIS

Aim: To determine the rate constant of the hydrolysis of an ester using an acid catalyst.

Principle: Hydrolysis of ester proceeds as

This reaction follows pseudo-first order kinetics.

The rate of the reaction is given by
Rate = k [Ester] [H₂O] [k = Constant]
Since water is present in large excess, rate of the reaction does not depend on concentration
of water and hence the rate become as
Rate = k′ [Ester] where k′ = k [H₂O]
The reaction can be followed by titrating aliquots of the reactions mixture with a given alkali
solution from time to time.If V0, Vt, and Vꝏ are volumes of alkali consumed at the beginning
of the reaction , after time t and after completion of the reaction respectively.
1 Vꝏ−V0
Then k′ = 𝑙𝑛 Vꝏ−Vt
𝑡
Vꝏ−V0
Rate constant is also determine graphically by plotting 𝑙𝑛 vs time (t).
Vꝏ−Vt

Apparatus required:
One 250 ml conical flask, 100 ml conical flask, pipette, burette, glass through.

Chemical required:
Stock solution of 0.1 N HCl solution, standardized NaOH solution (about 0.1 N), ethyl
acetate, phenolphthalein, distilled water.

Procedure:
1. Prepare 100 ml 0.1 N HCl: Take approximately 0.83 ml of conc. HCl in 100 ml conical
flask. Add 25 ml distilled water and mixed well to prepare homogeneous solution.
Adjust final volume of the solution 100 ml by adding distilled water up to mark.

35 | P a g e
 2. Prepare 250 ml 0.1 N NaOH: Take 1 g NaOH in a 250 ml conical flask. Add
approximately 50 ml distilled water and mixed well the solid to prepare
homogeneous solution. Adjust the final volume of the solution 250 ml by adding
distilled water up to mark.
3. Take 5 ml stock solution of ester added to this 100 ml 0.1 N HCl solution by pipette.
4. After 5 minutes take 5 ml of the above reaction mixture by pipette. Add to another
100 ml conical flask containing 20 ml of distilled water. Add few drops of
phenolphthalein indicator.
5. Titrate with 0.1 N NaOH solution and record the volume of NaOH that descending
from the burette immediately after appearance of pink colour.
6. Repeat the step 4 and 5, in every 5 minute calculate Vt according to these time
5,10,15,20 minute.
7. The volume of NaOH consumed in this titration is taken as V0.
8. To calculate Vo value, take 5 ml of the reaction mixture step 3 in a test tube. Its
mouth is loosely closed and then it is kept in a water bath for about 45 minute. Then
the content of the test tube is transferred into a conical flask: test tube is washed
and washing is also collected and it is titrated by same procedure of step 5. This
titre-value is Vo.

Calculation:
1. Arrange the result according to the following table :

Sl. No. Time Vol. of NaOH (Vꝏ – Vt) ln (V∞ – Vt) k′ =

1 Vꝏ−V0
𝑙𝑛 Vꝏ−Vt
(min.) (ml) (ml) (ml) 𝑡

(min-1)

1. 0

2. 5

3. 10

4. 15

5. 20

36 | P a g e
 6. 25

7. o

Vꝏ−V0
8. Plot a graph 𝑙𝑛 Vꝏ−Vt vs t:

Vꝏ−V0
𝑙𝑛 Vꝏ−Vt slope ( k′)

From the slope determined the rate constant.

Result: The rate constant for hydrolysis of an ester from

1. Calculated Value =
2. Graphical Value =

Questions:

1. What is meant by pseudo-first order reaction?

2. Difference between pseudo-first order and first order reaction?
3. Plot ln(V-V0)/(V-Vt) vs t. What was the rate constsnt for ester hydrolysis?

37 | P a g e
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