Detection of Extra Elements

DETECTION OF EXTRA ELEMENTS

Introduction

All organic compounds contain carbon, hydrogen and/ or oxygen. Some of them also contain elements like nitrogen, sulphur, chlorine, bromine, iodine, phosphorus etc. The elements other than carbon, hydrogen and oxygen are known as extra elements.

Detection of the extra elements is an important step in the investigation of an unknown organic compound. Hence it is significant to learn the methods used for identification of these elements in the laboratory.Since most of the organic compounds are covalent in nature. These extra elements are also covalently bound in the compound. Very few direct tests are known for their identification and hence these are first converted into ionic salts and then tested. The widely used method is that introduced by J.L. Lassaigne in 1843. It is known as the Lassaigne’s test.

Methods used for detection of extra elements present in the organic compoundsVarious methods used for detection of extra elements in the organic compounds are:

1. Lassaigne’s test for N, S and halogens2. Beilstein test for halogens

Theory

Lassaigne’s test

The principle of this test is that on fusion with sodium, the elements present in the organic compounds are converted to the corresponding ionic sodium salts. Thus, nitrogen in presence of carbon gets converted to cyanide ions, sulphur to sulphide ions and halogens to halide ions.

Organic compound containing C, N, S, X + Na NaCN+Na2S+NaX (X = Cl, Br, I)

The aqueous extract obtained after (i) fusing the organic compound with sodium and (ii) subsequent transfer of fused mass into distilled water, is called the Lassaigne’s extract or sodium extract, which is used for testing the presence of extra elements in an organic compound. The extract is alkaline in nature because of the presence of sodium hydroxide in it. Sodium hydroxide is formed by the reaction of hydrogen and oxygen (present in the organic compound) with sodium during fusion or/and because of the reaction of excess sodium (which has not reacted with organic compound) with water during the preparation of Lassaigne’s extract.

Test for nitrogenNitrogen present in the organic compound gets converted to sodium cyanide. It is tested by adding ferrous sulphate (part of which undergoes aerial oxidation to form ferric sulphate on heating) to the Lassaigne’s extract , heating and acidification with dilute sulphuric acid. Appearance of Prussian blue precipitate or blue or green colour (due to the formation of ferriferrocyanide) confirms the presence of nitrogen as an extra element in the organic compound.

Test for sulphurThe sodium sulphide formed due to the presence of sulphur in the organic compound can be tested by any of the following tests:(a) The Lassaigne’s extract is treated with sodium nitroprusside. A purple-violet

coloration appears when sulphur is present.Na2S + Na2[Fe(CN)5NO] Na4[Fe(CN)5NOS]

Purple-violet color

(b) The Lassaigne’s extract is acidified with acetic acid and then treated with lead acetate solution. Formation of black precipitate indicates the presence of sulphur.

Na2S + (CH3COO)2Pb PbS + 2CH3COONa Black ppt.

Test for N and S present together

If both N and S are present in the organic compound and enough sodium is used for fusion, a mixture of NaCN and Na2S are formed which are tested as above.

But if excess of sodium is not used for fusion or the fusion is incomplete, Na, C, N and S react to form sodium sulphocyanide (NaSCN) which is tested by acidification of the Lassaigne’s extract and addition of FeCl3

solution (Leibig’s test), a blood red coloration is obtained.N + S + C + Na NaSCN

3NaSCN + FeCl3 [Fe (SCN)3] + 3NaClFerric sulfocyanide Blood red

Formation of sodium sulphocyanide can be prevented by using slight excess of sodium. In such a case sodium cyanide and sodium sulphide are formed.

NaSCN + 2Na NaCN + Na2S

Test for halogensSilver nitrate test The Lassaigne’s extract contains NaCl, NaBr or NaI or a mixture of these, if one or more halogens are present in the organic compound.

In absence of N or S, the Lassaigne’s extract is acidified with dil. HNO3 and treated with AgNO3 solution. NaX + AgNO3 ——→ AgX↓ + NaNO3

A white precipitate, soluble in ammonium hydroxide is due to NaCl in the extract and Cl in the compound.

AgCl + 2NH3 ——→ [Ag(NH3)2]Cl (white ppt.)

A yellowish precipitate, sparingly soluble in ammonium hydroxide is due to NaBr in the extract and Br in the compound.AgBr + 2NH3 (excess) ——→ [Ag(NH3)2]Br

(pale yellow ppt.)

A yellowish precipitate, insoluble in excess of ammonium hydroxide is because of NaI in the extract and indicates I in the compound.AgI + NH3——→ Insoluble

(yellow ppt.)

If N or/and S are indicated in the compound, silver nitrate test is performed after removing NaCN as HCN and Na2S as H2S. This is achieved by acidifying the extract with dil. HNO3and boiling the resultant solution.

NaCN + HNO3 NaNO3 + HCN↑

Na2S + 2HNO3 2NaNO3 + H2S↑

It is essential to remove NaCN and Na2S from the extract before adding silver nitrate as NaCN and Na2S, if not removed form a white precipitate of silver cyanide and a black precipitate of silver sulphide respectively, thereby, interfering in the silver nitrate test for halogens.

NaCN + AgNO3 AgCN↓ + NaNO3

(white ppt.)Na2S + 2AgNO3 Ag2S↓ + 2NaNO3

(black ppt.)

Layer test for iodine and brominePositive silver nitrate test indicates the presence of halogens in the compound but which halogen is present is confirmed by the layer test.

In the layer test, the halide in the aqueous Lassaigne’s extract is oxidized to the corresponding halogen by KMnO4 or HNO3 or Cl2 water used as the oxidizing agent. The formed halogen is then extracted from the aqueous layer to the organic layer by the addition of CHCl3, CS2 or CCl4. The color in the organic layer indicates which halogen is present in the organic compound. A purple or violet color is due to iodine, an orange-brown color is due to bromine and no color (but a positive silver nitrate test) due to chlorine.

NaBr + H+ ——→ HBr + Na+

2HBr + (O) ——→ Br2 + H2O (soluble in CCl4)

NaI + H+ ——→ HI+ Na+

2HI + (O) ——→ I2 + H2O (soluble in CCl4)

Experiment

Aim: Detection of extra elements in the organic compound.

Learning Objectives: After performing the experiment, the student should be able to do the following:

1. Explain the term extra elements, and also the chemical reactions of the test carried out for detection of these elements.

2. Handle and dispose sodium (a highly reactive element) safely.3. Should be able to detect extra elements in any unknown organic

compounds.

Requirements

ApparatusIgnition tubes, a pair of tongs, china dish, wire gauze, funnel, test tubes, etc.

ChemicalsOrganic compound, sodium metal, ferrous sulphate, sulphuric acid, sodium nitroprusside, lead acetate, acetic acid, ferric chloride, nitric acid, silver nitrate, ammonium hydroxide, potassium permanganate, chlorine water chloroform or carbon tetrachloride.

Procedure

Preparation of Lassaigne’s extract1. Take a small piece ( 50mg) of freshly cut sodium (not yellow portion but

shinning silver surface).2. Dry it quickly by pressing it between the folds of filter paper and put it into a

clean dry ignition tube. Use a test tube in place of ignition tubes to prepare the sodium extract of a liquid compound.

3. Heat the tube slowly over a small flame until sodium melts (shiny round ball is formed).

4. Then remove the tube from over the flame and carefully add a small amount (~ 10-20mg or a drop) of the organic compound onto the molten sodium.

5. Heat the tube on a low flame at first and then strongly till it becomes red hot.

6. Keep the tube in the red hot condition for 1-2 minutes. While holding the tube in a vertical position, plunge it into about 10-12mL of distilled water contained in a china dish. Use a clean wire gauze while immersing red hot ignition tube in distilled water for protection in case of any spuriting or fire as unreacted sodium (if any) reacts violently with water.

7. Repeat such fusions 2-3 times and plunge the ignition tubes in the above china dish. The tube will be shattered and unreacted sodium will react with water.

8. Boil the contents of the china dish over a wire-gauze. 9. Filter the solution to get a clear alkaline filtrate (preferably colourless) called

Lassaigne’s extract. 10.Perform the tests for various extra elements using this solution.

Notes:1. The liquid compound vaporizes easily on heating in an ignition tube and hence

may not react with sodium. If heated in a test tube, the vapours of the organic compound condense on the cooler upper parts of the tube while heating and drop back on sodium. The liquid does not evaporate so easily and can hence react with sodium.

2. The shining piece of sodium should be cut and taken in each ignition tube at the time of fusion only. The simultaneous addition of sodium in all the ignition tubes should not be done as sodium reacts with atmospheric oxygen forming the oxide layer over the metal and hindering its reaction with the organic compound.

3. Certain compounds (e.g. azo and polynitro compounds) react explosively when heated with sodium.

4. There should be no unreacted compound at the end of the fusion. The unreacted compound may cause the following problems during the testing of extra elements;(a) The extract may be coloured (phenols, carbohydrates may give a coloured

extract).(b)The unreacted compound may remain dissolved in the alkaline extract and

get precipitated on acidification of the extract. Thus interfering in the test for extra elements (Acidic compounds may cause this problem).

Test for Nitrogen1. To approximately 1mL of Lassaigne’s extract in a test tube, add about 1mL of

freshly prepared ferrous sulphate solution or a few crystals of ferrous sulphate. 2. Boil the solution gently.3. Cool and acidify with dilute sulphuric acid.

4. A Prussian blue, blue or green colour confirms the presence of cyanide ion in the solution and hence nitrogen in the given compound.

Note: 1. Hydrochloric acid should not be used for acidifying the alkaline solution

since the yellow colour, due to the ferric chloride formed, causes the Prussian blue to appear greenish. For the same reason, ferric chloride should not be added as is frequently recommended; a sufficient concentration of ferric ions is produced by the atmospheric oxidation of the hot alkaline solution of ferrous salt.

2. Nitric acid should not be used for the acidification of extract as it will oxidize all Fe2+ to Fe3+ completely and a Prussian blue colour will not be obtained.

3. The extract should be boiled gently for a few seconds after the addition of ferrous sulphate as excessive boiling may cause total conversion of Fe2+ to Fe3+.

Test for Sulphur(a) Nitroprusside test:

1. Take approximately 1mL of Lassaigne’s extract in a test tube.2. Add a few drops of freshly prepared aqueous sodium nitroprusside solution

or a few crystals of sodium nitroprusside. 3. A violet or a purple colour confirms the presence of sulphide ion and hence

sulphur in the compound.(b) Lead acetate test:

1. Acidify about 1mL of Lassaigne’s extract with acetic acid in a test tube.2. Add into it about 1mL of lead acetate solution. 3. The appearance of black precipitate indicates sulphide ion in the solution

and hence sulphur in the given compound.

Note: If the extract is not acidified before adding lead acetate, a precipitate of lead hydroxide is formed on the addition of lead acetate.

Test for nitrogen and sulphur present togetherLeibig test: This test is positive if NaSCN is formed on the fusion of the compound containing both N and S with sodium.

1. Acidify approximately 1mL of Lassaigne’s extract with dilute HCl in test tube.

2. Add into it about 1mL of ferric chloride solution. 3. The appearance of a blood red colour indicates the presence of thiocyanate

ion in solution and hence nitrogen and sulphur both present in the given compound.

Note: If excess of sodium is used for the fusion during the preparation of Lassaigne’s extract, NaCN and Na2S are formed (instead of NaSCN) which can be tested as above.

Test for halogens

(a) Silver nitrate test with Lassaigne’s extract:If Nitrogen and/or Sulphur are absent: 1. Take approximately 1mL of Lassaigne’s extract in a test tube.2. Acidify with dilute nitric acid.3. Add about 1mL silver nitrate solution (Dilute nitric acid is added to

neutralize sodium hydroxide otherwise a brown precipitate of silver hydroxide is obtained).

4. (i) A white precipitate readily soluble in aqueous ammonia solution confirms chlorine.

(ii)A pale yellow precipitate partially soluble in aqueous ammonia solution confirms bromine.

(iii) A yellow precipitate insoluble in aqueous ammonia solution confirms iodine in the given compound.

If Nitrogen and/or Sulphur are present:1. Take approximately 2mL of Lassaigne’s extract.2. Add 1mL concentrated nitric acid and boil to half its original volume. 3. Cool it and then proceed as above (Steps 3 and 4 of above procedure).

Note:1. In case of positive test for halogens, perform a blank test as distilled water

used may be contaminated with chloride ions as impurity. To perform the blank test, use distilled water in place of Lassaigne’s extract and proceed as above. A white precipitate indicates the presence of chloride ions in the distilled water.

2. Use chloride free distilled water for the tests.3. Excess sodium in solution forms sodium hydroxide with water, which reacts

with silver nitrate solution to form a brown precipitate of silver hydroxide.

As dilute nitric acid neutralizes sodium hydroxide, it should be added before silver nitrate solution in test for halogens.

4. N and S in the organic compound interfere in the silver nitrate test for halogens and hence these must be completely removed before adding silver nitrate to the Lassaigne’s extract.

(b) Layer test (for bromine and iodine): 1. Acidify approximately 1mL of Lassaigne’s extract with conc. nitric acid in a

test tube.2. Gently boil the solution.3. Add 1mL of organic solvent (carbon tetrachloride or chloroform) and shake

vigorously.4. (i) Orangish brown/yellowish orange colour in organic layer confirms bromine.

(ii)Violet/purple colour in organic layer confirms iodine in the given compound.

Note1. Potassium permanganate or chlorine water may be used as oxidizing agent

instead of conc. Nitric acid.2. The extract should not be boiled excessively with the oxidizing agent as the

formed halogen may escape during heating.Sources of error: Impure sample. Distilled water itself contains chloride ions. Fusion of the given organic compound with sodium metal not done properly. Presence of excess of organic sample and not sufficient amount of sodium

present to react.

Precautions:(1)Safety glasses must be worn.(2)Sodium must be kept away from water.(3)Organic compound must be added enough to react with whole of sodium in the

fusion/ignition tube.(4)Excess of the organic compound must be burnt completely before plunging the

tube into distilled water, otherwise the extract may be coloured (which can not be used for the detection of extra elements).

(5)Heating must be done over a small flame initially so that the organic compound does not get volatilized.

(6)Distilled water must be used for preparation of Lassaigne’s extract.

(7)A wire gauze should be used while immersing red hot ignition tube in distilled water contained in a china dish for protection against mishap.

(8)A freshly prepared solution of sodium nitroprusside and ferrous sulphate should be used.

(9)The unused cut pieces of sodium should be put back into the bottle containing paraffin or kerosene oil and should never be disposed in the dustbin or sink. The small pieces of sodium which are taken in the ignition tube for fusion with the organic compound (not fused with the compound because (i) either the ignition tube got cracked while heating or (ii) was not used for fusion) should be placed in a dry test tube and treated with a small amount of ethanol

Questions for viva-voce:1. What are organic compounds? Name the elements essentially present in all

organic compounds.2. What do you understand by the term extra elements? Give a few examples.3. What is Lassaigne’s extract and how is it prepared?4. Why is it essential to prepare Lassaigne’s extract for the detection of extra

elements in the organic compound?5. Why is Lassaigne’s extract alkaline?6. Give the chemistry of Lassaigne’s test of Nitrogen.7. Why ferric chloride should not be added in the test of nitrogen?8. Give the formulae of the complex responsible for Prussian blue colour in test

for nitrogen.9. Why nitric acid and hydrochloric acid can not be used for acidification of the

extract in the test of nitrogen?10.Explain why inspite of the presence of nitrogen in the following compounds,

their Lassaigne’s extract gives negative test for nitrogen: Hydroxyl amine, hydrazine, ammonium chloride and ammonium sulphate.

11.Discuss the chemistry of Lassaigne’s test for sulphur.12.Can sulphuric acid be used instead of acetic acid for the acidification of

Lassaigne’s extract for lead acetate test for sulphur?13.Why is it necessary to acidify the Lassaigne’s extract for performing the

following tests: (a) Lead acetate test for sulphur (b)Silver nitrate test for halogens(c) Test for nitrogen?

14.What products are formed when the organic compound containing both nitrogen and sulphur is fused with:

(a) excess of sodium(b) limited amount of sodium?

15.Discus the chemistry of silver nitrate test for halogens.16.Why is it necessary to remove N and/or S if present before testing halide ions?17.A white precipitate of silver chloride dissolves in ammonia solution. Why? On

addition of dilute nitric acid, white precipitate reappears. Explain.18.Can sulphuric acid or hydrochloric acid be used for the acidification of

Lassaigne’s extract for silver nitrate test for halogens?19.Discuss the chemistry of the Layer’s test giving chemical equations.20.Why is it recommended to do blank test for confirming the presence of chlorine

as extra element in the given compound?21.Which functional groups are possible if a) nitrogen, b) sulphur, c) halogen, and

d) nitrogen and sulphur together are present as extra elements?

Sample set of observationLassaigne’s Test for Nitrogen, Sulphur and Halogens:

S. No. Experiment Observation Inference 1.

Test for nitrogenA few crystals of ferrous sulphate or 1mL of freshly prepared ferrous sulphate solution was added to 1mL of Lassaigne’s extract in a test tube and the mixture was boiled gently.

A green precipitate was formed.

Dilute sulphuric acid was added to the precipitate.

OR

A Prussian blue or a deep green coloration observed.

Cyanide ion present and hence nitrogen is present in the given organic compound.

Dilute sulphuric acid was added to the precipitate.

No blue coloration or ppt. is obtained

Nirogen absent

2.

Test for sulphurNitroprusside test:

1 mL of freshly prepared sodium nitroprusside solution (prepared by dissolving a crystal of sodium nitroprusside in 1mL of distilled water) was added to the 1mL Lassaigne’s extract in a test tube.

OR

Intense violet colouration observed.

Sulphide ions present and hence sulphur is present in the given organic compound.

1 mL of freshly prepared sodium nitroprusside solution (prepared by dissolving a crystal of sodium nitroprusside in 1mL of distilled water) was added to the 1mL Lassaigne’s extract in a test tube.

No violet colour observed.

Sulphur absent.

Lead acetate test:Acidified 1mL of Lassaigne’s extract with acetic acid in a test tube and then added 1mL of lead acetate solution.

OR

A black precipitate was formed.

Sulphide ions present and hence sulphur is present in the given organic compound.

1 mL of freshly prepared sodium nitroprusside solution (prepared by dissolving a crystal of sodium nitroprusside in 1mL of distilled water) was added to the 1mL Lassaigne’s extract in a test tube.

No black precipitate formed.

Sulphur absent.

3.

Test for nitrogen and sulphur if present together:Leibig test:Acidified 1mL of Lassaigne’s extract with dilute HCl in a test tube and then added about 1mL of ferric chloride solution.

OR

A blood red colouration was observed.

Nitrogen and sulphur both present in the given compound indicating incomplete fusion

Acidified 1mL of Lassaigne’s extract with dilute HCl in a test tube and then added about 1mL of ferric chloride solution.

No blood red coloration observed.

Both Nitogen and sulphur absent.

4.

Test for halogens:Beilstein test:A copper wire was heated till a green flame was no longer seen. This wire was then dipped in the compound and heated in the upper part of the flame.

Bright green flame was observed.

Halogen(Cl, Br or I) present

AgNO3 test:Acidified the 1mL of Lassaigne’s extract with dilute nitric acid in a test tube. Then added 1mL of silver nitrate solution (if nitrogen and /or Sulphur is/are present and dilute nitric acid, boiled the solution and then added silver nitrate solution).

a) white precipitate obtained which is soluble in ammonia solution

ORb) the pale yellow

precipitate obtained which is partially soluble in ammonia solution

ORc) deep yellow

precipitate obtained which is insoluble in ammonia

a) Chlorine is present.

b) Bromine is present.

c) Iodine is present.

Layer test:To 1mL of Lassaigne’s solution added 1 mL of concentrated

a) A colorless layer was observed.

a) Chlorine may be present or halogens are

HNO3; warm gently (to oxidize the halide ion to the respective halogen gas which dissolves in the organic layer) add 1mL of carbon tetrachloride or chloroform and shake well.

b) Yellowish-orange color observed in the organic layer

c) Violet colour observed in the organic layer

absent.b) Bromine is

present.

c) Iodine is present.

Beilstein test for halogens

IntroductionThis is a preliminary general test which may be used for the detection of a halogen (Cl, Br or I) in an organic compound. The nature of the halogen (Cl, Br or I) cannot be confirmed by this test. For confirmation, the Lassaigne’s test (Silver nitrate test and Layer test) has to be performed. In this test, a copper wire is dipped in the organic compound and heated. A green coloured flame indicates the presence of halogens.

TheoryThis test depends on the fact that the hot copper wire has a layer of copper oxide, which reacts with halogen present in the organic compound forming corresponding copper halide, which, being volatile at high temperatures, gives the usual green copper colouration. The importance of this test is that it is unaffected by the presence of nitrogen, and thus gives a rapid indication of the definite absence or probable presence of halogen in the organic compound

Procedure1. Heat a copper wire till no more green flame is seen. 2. When still hot, expose it to the organic compound and heat it again.3. A bright green flame, often lasting for a few seconds, indicates presence of

halogens in the organic compound.

Note: 1. In the Beilstein test for halogens, compounds like urea, thiourea, and

benzamide interfear because these also give a green flame because they form volatile compounds with copper.

2. If the compound is a polychloroarene, the highly toxic chloro-dioxins may be forme during the flame test and hence the test is not popular.

PURIFICATION OF THE ORGANIC COMPOUNDS CRYSTALLISATION

Introduction

An organic compound which is either synthesized in the laboratory or isolated from any natural source is rarely obtained in a pure form. It is invariably contaminated with other compounds i.e. impurities. Purification of the compounds thus becomes very important, for its identification as the pure compound has a sharp and well-defined melting point and for its final use as a pharmaceutical, a food colorant, flavour, perfume, pesticide, fertilizer etc.

A number of methods have been used for the purification of the compounds. For example, a solid compound may be purified by one or a combination of the follwing methods:

Crystallization Fractional Crystallization Sublimation Solvent extraction Chromatography etc.

Theory

Purification by crystallizationThe principle underlying crystallization is that the solubility of a compound in a solvent increases on increasing the temperature and vice-versa. So when a saturated solution of a compound is prepared in a suitable solvent at high temperature (i.e. boiling point of the solvent), the amount of solute dissolved is much more as compared to what would have dissolved at room temperature or a lower temperature, so when a hot solution is allowed to cool, the extra or excess solute that is dissolved separates or crystallizes out and can be obtained by filtration.

This technique is used as a simple method for purification of compounds. The impure compound is dissolved in a solvent such that the impurities are either insoluble even in the hot solvent or so soluble that these remain dissolved even at a lower temperature.

Hence for purification of a solid compound by crystallization, the choice of the solvent is very important.

The various steps involved in the crystallization of a solid compound:

Selection of a solvent:In order to select a solvent for purification of the compound, take about 50mg of the compound in a test tube ,add to it about 2-3ml of the solvent (water, methanol, ethanol, ethyl acetate etc. are some common organic solvents used for crystallization) and heat the test tube in a hot water bath containing inflammable liquids or directly on a flame if water is used as asolvent. To choose a suitable solvent, the following points should be kept in mind:

1. The solvent should be inert, that is, it should not react with the compound to be purified.

2. The compound to be purified should be highly soluble in the solvent at high temperatures and almost insoluble at room temperatures.

3. The impurities should be either soluble in the solvent at room temperature such that they remain in the mother liquor and do not crystallize out or are insoluble even at high temperatures and thus can be removed by hot filtration.

4. The boiling point of the solvent should be preferably lower than the melting point of the compound.

5. The rule of ‘like dissolves like’ should be kept in mind while selecting a solvent.

Note:If a single solvent does not meet the above criteria, a mixture of solvents (which are miscible with each other e.g water-ethanol, petroleum ether-ethyl acetate, etc.) may be used for crystallization.

Dissolution of the solute in the required amount of solvent: Take the impure solid in a clean test tube or a boiling tube. Add a few milliliters (4-5) of the solvent and heat the solution directly on a flame with water as solvent

or in case an inflammable organic solvent is being used as solvent, heat the solution on a water bath with constant stirring using a glass rod. If the solid does not dissolve, add a few milliliters of the solvent and heat again (do not add excess of solvent). If near the end of the dissolution process, it appears that an additional amount of the solvent is not dissolving any more of the solid, then this small amount of undissolved solid is likely to be an insoluble impurity. Do not add any more solvent but filter the hot solution through a fluted filter paper (Fig. 1).

Fig. 1: How to fold to get a fluted filter paper.

Note:If a mixture of two solvents is used for crystallization-dissolve the solute in a minimum amount of the solvent in which it is more soluble and then add the other solvent (in which the compound is insoluble or less soluble) dropwise till turbidity just appears. Heat the turbid solution directly on a flame or a hot water bath (if inflammable solvents are being used) to obtain a clear solution and keep aside for crystallization.

Decolourization:If the solution obtained is highly coloured, add a small amount of animal charcoal, boil the solution for some time. (The coloured impurities are adsorbed on the surface of charcoal.) Filter the hot solution through a fluted filter paper and allow the filtrate to cool for the separation of crystals.

Crystallization:Do not cool the solution obtained above rapidly, allow it to slowly cool to room temperature. If no crystals are obtained, cool the solution in an ice-bath or scratch the sides of the test tube with a glass rod, just near the surface of the solution to

initiate crystallization or add 2-3 crystals of the solid crude compound to induce crystal formation.

Filtration and drying:Filter the crystals by vacuum filtration and collect the mother liquor in a clean dry boiling tube. Cool the mother liquor in an ice bath, concentrate and collect to obtain the second crop of crystals. Filter and air dry or keep in a vacuum desiccator.

Some important terms involved are:Crystallization point:The stage at which solution becomes saturated with respect to the solute is called crystallization point.

Mother liquor:The residual fluid filtrate left behind after the separation of crystals from the saturated solution is called mother liquor.

Bumping:It is the spurting out of the solution while being heated. To some extent, bumping can be reduced by adding two or three small pieces of pumice stone to the solution being heated.

Seeding:Sometimes, no crystals are obtained on cooling the solution even though it is concentrated enough. Then during the cooling and scratching, a minute quantity of the crude material may be added to “seed” the solution. It is called “seeding” and it facilitates initial crystallization.

Aim: Purification of the given compound by crystallization using:(i) water(ii) alcohol as the solvent.

Learning Objectives: After performing the experiment, a student:

(i) learns the importance of purification of the compound.(ii) can choose an appropriate solvent for crystallization of unknown

compound.

(iii) learns that on the basis of difference in solubilities, the impurities can be removed to obtain pure compound.

(iv) learns to handle and heat inflammable organic solvents.(v) learns to make a saturated solution.(vi) learns to make a fluted filter paper.(vii) learns the use of vacuum pump.(viii)learns about various filtration apparatus used.

Requirements:

ApparatusTest tubes, boiling tubes, funnel, filtering tube, filter button, Buchner funnel, Buchner flask, tongs, glass rod, etc.

ChemicalsGiven solid compound, alcohol and charcoal.

Procedure

Crystallization using water as solvent:1. Take ~0.5g of the impure compound in a boiling tube and add minimum

amount of water (about 5mL in the beginning).2. Heat the contents to boiling by holding the boiling tube with a test tube

holder (stir while heating using a glass rod). Add more hot water if required. 3. Remove the suspended impurities by filtration of the hot solution. To carry

out hot filtration, filter the boiling hot solution through a fluted filter paper in a pre-heated funnel. Collect the filtrate in a pre-heated boiling tube (carry out the pre-heating by heating the boiling tube, funnel and fluted filter paper in an oven).

4. Allow the filtrate to cool slowly in air to room temperature.

Crystallization using alcohol as solvent:1. Take 0.5g of the impure compound in a boiling tube and add minimum

amount of alcohol (about 5mL in the beginning). 2. Heat the contents to the boiling point of the alcohol in a boiling water bath.

Add more hot alcohol, if required, to dissolve the compound. Then follow step 3 and 4 as above.

Filtration: Using a filtering tube and a funnel(i) Take a rubber cork to fit into the filtering tube.(ii) Make a hole in the centre of the cork and fit a filter funnel into it, the

fitting should be air-tight.(iii) Fix the cork with the funnel in the filtering tube.(iv) Clamp it near the filtration pump.(v) Fix a filter button or a small filter disc to cover the stem of the funnel.(vi) Cut a round piece of filter paper (slightly bigger than the button or disc).(vii) Wet the filter paper with water. Place it on the button or filter disc.(viii) Connect the side tube of the filtering tube to the vacuum pump.(ix) Switch on the vacuum pump.(x) Transfer the crystals along with mother liquor on the funnel.(xi) Allow the mother liquor to drain completely.(xii) Disconnect the connecting tube from the pump.(xiii)Transfer the crystals from funnel to a filter paper. (xiv)Allow to dry in air.

Note:A Buchner funnel and a Buchner flask may be used instead of a funnel and a filtering tube for filtration if the amount of the solid is more.

Filtering tube Buchner flask and funnel

Vacuum pump

Fig. : Vacuum filtration assembly

Precautions1. Do not add excess of the solvent for the dissolution of the solute.2. While dissolving the impure sample in an inflammable solvent like alcohol,

do not heat the test tube directly on a flame but heat it in a hot water bath. (Either clamp it or hold with a test tube holder)

3. Add a few pieces of pumice stones before heating to avoid bumping. 4. Do not take alcohol near the flame. 5. Allow the solution to cool slowly for crystallization and do not disturb it

during the crystallization process.6. Spread the crystals on the filter paper for drying.7. Do not crush the crystals while drying.

ResultTotal yield of the crystallized organic sample obtained =

Note: Prepare a hot saturated solution of the compound for crystallization. Add pumice stones before heating the solution and never while heating. In case of purification of a compound which is highly insoluble in the

solvent at room temperature, a slight excess of the solvent is required. But in other cases especially while using alcohol as solvent, one must use minimum amount of the solvent.

In case no crystals appear even after keeping the filtrate at room temperature, it may be due to the fact that filtrate is not saturated. So concentrate it to reduce the volume by heating it directly over flame in case

of water as solvent and by heating it in water bath in case of alcohol as solvent. Scratch the sides of the test tube with a glass rod, just near the surface of the solution to initiate crystallization.

To ensure the purity of crystallized sample, determine its melting point. In general it may be said that a pure compound has usually a sharp melting point i.e. the substance melts entirely within a range of about 1ºC, whereas an impure substance does not have a sharp melting point and will therefore melt slowly and indecisively over a range of several degrees.

Questions for viva-voce:1. Describe the principle behind crystallization.2. Discuss the various steps involved in crystallization.3. What is fluted filter paper and how is it made?4. How to select a suitable solvent for crystallization of an organic compound?5. Why is it necessary to use pumice stone while heating the solution?6. How are the soluble coloured impurities removed?7. What is the advantage of vacuum filtration over normal filtration using

funnel and filter paper?8. What is ‘mother liquor’?

9. Name some solvents which can be used for crystallization.

DETERMINATION OF PHYSICAL CONSTANTS

Melting point

Introduction

The melting point of a crystalline solid is the temperature at which it begins to change into a liquid under a pressure of one atmosphere. Melting point of a compound is depressed (lowered) due to the presence of impurities. Melting point is an important property of a compound, It is determined to know whether the compound is pure or not. A pure compound has a sharp melting point whereas an impure sample melts over a range of temperature. It is an important physical property and hence is helpful in characterization of an unknown compound. Melting point and mixed melting point give useful information about the identity

and non identity of two compounds. If the mixed melting point of two compounds is same as that of the individual compounds, the compounds are identical otherwise they are non identical. Methods used for determination of melting point

Using Kjeldahl’s flask Electrically heated melting point apparatus

Theory

Criteria of purity: solid compounds

The property of an organic compound which is most frequently determined as a criterion of purity is its melting point, because in general it may be said that a pure compound has a sharp melting point i.e. the substance melts entirely within a range of about 1°C, whereas an impure substance has an indefinite melting point and therefore melts slowly and indecisively over a range of several degrees.

The melting point of a given sample may or may not be sharp. The reasons could be:i. Sharp melting point(a) The substance is chemically pure. This is almost invariably the cause of a sharp melting point.(b) The substance is a eutectic mixture of two or more compounds-a rare possibility.

(ii) Indefinite Melting point:a) The substance is impure. This is almost invariably the cause of an indefinite melting point.b) The substance is pure, but on warming undergoes slight thermal decomposition before the melting point is reached and the decomposition products then acts as impurities and depress the melting point.

Mixed melting point:The mixed melting is determined to establish the purity of known organic compounds or to show the identity or non-identity of two compounds. In this technique the compound under investigation is mixed with a little amount of the same pure compound or the two compounds, the identity of which is to be

established, are mixed together and powdered. The mixture so obtained is filled in the capillary and the melting point determined in the usual manner. If the value of the melting point of the mixture is lower than that of pure compound then the compound under investigation is impure (or the two compounds are non-identical) but if the melting point of the mixture is same as that of the pure compound then the compound under investigation is pure (or the two compounds are identical).

Use of Kjeldahl’s flask for the determination of the melting point:

Cleaning and drying of Kjeldahl flask1. Clean the flask first with a detergent (never use chromic acid for cleaning) and

wash with water.2. Keep the clean flask inverted on a tripod stand so that all water drains out.3. Rinse the flask with acetone and again invert it on a tripod stand to drain all

the acetone.4. The flask is almost dry.5. keep the Kjeldahl flask in a hot oven for 5-10 minutes.6. Take out the flask and allow it to cool to room temperature.7. If any moisture condenses on the inner sides of the flask on cooling, heat it in

the oven again for about 5 minutes otherwise use the flask for melting point determination.

After drying, clamp the flask and take one of the following bath liquid in it ( fill about half of the bulb)

Bath liquidsGenerally three liquid compounds (i) concentrated sulphuric acid (ii) medicinal paraffin (iii) silicone oil (marketed as MS550 fluid) are be used as bath liquids in Kjeldahl’s flask for the determination of melting point.

Conc. Sulfuric acid is one of the best bath liquids though it is highly corrosive in nature.

Medicinal paraffin It has a low specific heat and therefore the temperature can be increased using only a small flame.

1. Even when hot it is almost non-inflammable and therefore should the flask break whilst still over the flame, the oil seldom ignites.

2. The oil is non-corrosive, and owing to its low specific heat causes remarkably slight burns even if spill while at a high temperature on the hands.

3. The oil may be safely heated up to about 2200C, when it begins to decompose slightly, giving off smoky fumes.

Silicone oilSilicone oil (marketed as MS550 fluid) can be used in place of sulphuric acid and paraffin oil for compounds of high melting points. It is a straw-coloured non-corrosive liquid which can be safely heated upto 3000C without decomposition or ignition.

Checking the calibration of the thermometer

A 360ºC thermometer is normally used for determination of the melting point. The least count generally is 1ºC.

The thermometer is calibrated by the manufacturer. For marking the thermometer, one of the procedure adopted is- The thermometer is kept in ice (for 0ºC mark) and in boiling water (for 100ºC mark) and then marked for rest of the temperatures up to 100ºC by dividing the length (between 0ºC and 100ºC mark) equally into 100. Similarly the thermometer is marked for temperatures from 100-360ºC.

The calibration of the thermometer should be rechecked before using it for determination of the melting point of an unknown compound.

If the calibration is not checked, a thermometer may give wrong results because of one or more of the following reasons:1. The capillary into which mercury rises to indicate the temperature may not

have a uniform hole size.2. The mercury thread or column breaks while determining the melting point i.e.

air space is formed in the mercury thread. (Air and mercury have different expansion coefficients.)

3. The markings may not have been correctly done.

For rechecking the calibration of the thermometer, determine the melting point of a few standard compounds following the procedure given below. Some of the standard compounds which can be used are:

p-Nitrotoluene (54ºC), Benzil (95ºC), oxalic acid dihydrate (101ºC), benzoic acid (122 ºC), salicylic acid (156 ºC), succinic acid (185ºC) and 3,5-dinitro benzoic acid (205 ºC) and p-Nitro benzoic acid (239 ºC).

If the melting points of the standard compounds are as reported in the literature, the thermometer can be used for the melting point determination of any unknown compound.

Aim: Determination of the melting point of the given organic compound

Learning objectives: After performing the experiment, a student should know

What is melting point? What is the importance of correct melting point determination? What is the effect of impurities on the melting point of a compound? Meaning and the necessity of calibration of the thermometer How to handle a corrosive liquid like sulphuric acid The meaning and the significance of mixed melting point.

RequirementsApparatusKjeldahl’s flask, sand bath, clamp and stand, thermometer fitted in the cork, spatula, watch glass, a thin capillary tube and Bunsen burner.

Chemicals Given organic compound, concentrated sulphuric acid (or paraffin or silicone oil )

Procedure

1. Clean and dry the Kjeldahl’s flask (made of Pyrex glass) and fill about half the bulb of the flask with concentrated sulphuric acid (or any other bath liquid).

2. Clamp the flask about 12 inches from the base, vertically on a stand (Fig. ).

3. Lower the thermometer fitted with a grooved bark cork into the flask such that the bulb of the thermometer is completely immersed in the bath liquid (Fig. ) but is not touching the base.

4. Place a sand bath on the base of the stand beneath the Kjeldahl’s flask. 5. Powder the compound (100mg) well on a watch glass with the help of a

clean spatula.6. Seal one end of the capillary tube by inserting the end of the capillary tube

horizontally into the extreme edge of a small steady burner flame for a few seconds, rotating the capillary meanwhile.

7. Then pack the powdered compound (to form a compact column) into this capillary tube to a length of 3-5mm by pushing the open end of the capillary through the powder and tapping its closed end on the bench or by making it fall through a long glass tube on the table 2-3 times.

8. Wipe the capillary from outside to remove any adhering organic compound.9. Take out the thermometer dipped in concentrated sulphuric acid bath and

apply a few drops of it on the outer side of capillary tube containing the powdered compound. Attach the capillary to the lower end of the thermometer so that the substance is at the lower level of the thermometer bulb.

10.Lower the thermometer with the capillary tube into the sulphuric acid bath slowly and carefully so that the bulb of the thermometer and the part of the capillary tube (containing packed material) are well immersed in concentrated sulphuric acid. (The thermometer markings should face the observer.)

11.Heat the Kjeldahl’s flask by holding the burner in one hand. Rotate the burner constantly for uniform heating of the flask.DIAGRAM

12.Note the temperature at which the compound starts melting along with the temperature at which it completely melts (solid in the capillary tube changes into a transparent liquid). This gives the melting point of the solid.

13.Allow the flask and bath liquid to cool slowly in air. Never cool under a running tap water.

14.Repeat the melting point determination once more. 15.If the two observations are identical or vary by 1ºC, record the observation

otherwise repeat the experiment. 16.Remove the capillary from the bath after each melting point determination.

Note: 1. The compound used for melting point determination should be pure as only a

pure compound has a fixed and a sharp melting point

2. The capillary tube should be very thin walled, about 8cm long and 1mm in diameter.

3. If the markings are not clearly visible on the thermometer, rub some charcoal powder on the markings and wipe off the excess charcoal.

4. Use a non-luminous flame for heating.

Grooved cork

INCOMPLETE DIA.-----Sources of errors:

1. Capillary tube is not sealed properly so bath liquid enters the capillary tube.2. Thermometer is not calibrated.3. The capillary tube is too thick.4. The sample is not powdered well.5. The sample is not dry.6. The sample is not packed properly7. The sample is impure.8. The packed sample in the capillary tube is not near the bulb of the

thermometer.9. The heating is not uniform.10. Fast heating near the melting point gives incorrect results.11. Delay in noting down the temperature at which the sample melts.

Precautions: 1. Wear safety glasses.2. Always wear a cotton laboratory coat while working in the laboratory.3. Capillary tube breaks easily, hence handle it carefully.

4. Seal the capillary tube properly otherwise concentrated sulphuric acid would enter the capillary tube. It should not bend while sealing.

5. Crush the sample to a fine powder for filling it in the capillary.6. Keep a sand bath on the base of the clamp beneath the Kjeldahl flask so that

in case the flask breaks and sulphuric acid falls, it is soaked by the sand kept in sand bath and causes minimum damage to the person performing the experiment and also to the working table.

7. Clamp the Kjeldahl’s flask properly, neither too tight nor too loose.8. The bulb of the thermometer and the packed material in the capillary tube

should be well immersed in the bath liquid.9. The lower tip of the capillary tube should be at the same level as the bulb of

the thermometer and the capillary should be straight.10.Use a bark cork and not a rubber cork for holding the thermometer.11. Use a grooved cork for the thermometer.12.Rotate the burner throughout so that heating is uniform.13.Note the temperature at which the solid begins to melt (shrinks slightly) and

the temperature at which it melts completely.14.If the acid in the concentrated sulphuric acid bath becomes coloured through

contact with organic matter, it can be easily decolourized by adding a crystal of potassium nitrate and heating.

15.The rate of heating near the melting point should be very slow (not more than 1-2ºC rise in temperature per minute).

16.Avoid superheating.17.After determination of the melting point the acid bath should be allowed to

cool to room temperature.18.Sulphuric acid (though best as a bath liquid) is highly corrosive. It causes

severe skin burns and should be handled very carefully (See first aid instructions in the laboratory in case of any accident).

19.Adhere the capillary well to the thermometer so that it does not drop in the flask.

Alternative apparatus A boiling tube may be used instead of a Kjeldahl’s flask for holding the bath

liquid (Fig. )Note: The tube should be heated slowly using a small flame otherwise superheating can occur because of the much smaller amount of bath liquid.

A beaker may be used instead of a Kjeldahl’s flask (Fig. )

Fig. : Alternative apparatus used for determination of the melting point

Observations:Melting point of the given compound = Literature melting point of the sample = Reference for the literature m. pt.:

Result: The melting point of the given organic compound is found to be ……….

Questions for viva-voce:1. What is melting point?2. List out the possibilities of having sharp melting point.3. Name some of the chemicals which can be used as bath liquid in the melting

point determination.4. What are the advantages and disadvantages of using paraffin over

concentrated sulphuric acid as bath liquid?5. Why is it necessary to use grooved cork to fit the thermometer in the

Kjeldahl’s flask?6. Explain the criterion of purity in case of a solid organic compound.7. Why it is necessary to calibrate a thermometer?8. Name a few compounds used for checking the calibration of the

thermometer9. What do you mean by the literature melting point of the compound?10.Why is it advised to slow down heating near the melting point of the

compound?

11.Why is it necessary to keep a sand bath below the Kjeldahl’s flask while determining the m. pt.?

12.What is mixed melting point?13.What is the effect of impurities on the melting point?

Electrically heated melting point apparatus: The melting point can be determined by using an electrically heated melting point apparatus. The advantage of using it is that high temperatures can be reached with safety and the dangers of hot oil or concentrated sulphuric acid are avoided. The powdered sample is either filled in the capillary (as in the Kjeldahl’s method) or placed on a small glass plate and heated electrically in the melting point apparatus. The melting point is then recorded using a thermometer (Fig. ) or automatically (Fig. ).

Fig. no. --- Electrically heated melting point apparatus

Note: A rough idea of the melting point of an unknown compound should be obtained first and then the rate of heating should be controlled to get the accurate melting point of the compound.

Boiling point

Introduction

The boiling point of a liquid is the temperature at which its vapour pressure is equal to the atmospheric pressure and the liquid begins to change into the gaseous state. Boiling point is determined to know- weather the compound is pure or not.( A pure compound generally has a sharp boiling point whereas an impure sample boils over a range of temperatures). It is an important property of a compound and is helpful (like the melting point ) in the identification of a liquid organic compound. A given organic compound gets purified during distillation, when the boiling point is determined by distillation method Methods used for boiling point determination

Distillation method Kjeldahl’s method (Siwoloboff’s method)

Theory

Distillation method

A pure liquid (which distills without decomposition) will have a sharp boiling-point, the temperature remains constant until the whole of the liquid has boiled off, leaving no residue.Unlike the melting point, however, boiling point whilst remaining sharp, may vary in value over a range of several degrees, owing to the variation in the pressure. The boiling point of an impure liquid depends largely on the physical nature of the impurities. If all the impurities are non-volatile, the liquid will have a sharp boiling point, the non-volatile impurities remain as residue when the liquid has evaporated.If the impurities are volatile, then the boiling point of the liquid may remain constant, may rise steadily as the liquid boils, or may rise in a series of definite steps, according to the nature and quantity of the impurities present.Although a pure solid has a sharp melting point, the converse is not necessarily true; a sharp boiling point does not always indicate a pure liquid, but may be caused by a constant boiling mixture of two or more liquids. The method used for the determination of the boiling point depends upon the quantity of the liquid available for carrying out the determination. When a few milliliter of the liquid are available, the distillation method may be employed. When only a few drops of the liquid are available, Kjeldahl’s method is used.

Cleaning and drying of the apparatus used for distillation for determination of the boiling point Two types of apparatus may be used for determination of the boiling point

1) Glass apparatus with interchangeable joints

2) Glass apparatus assembled using bark corks for assemblyQuick-Fit Apparatus-Glass apparatus with interchangeable (I/C) joints The Quick-Fit apparatus has joints, which fit with each other. No connecting corks are required. The joints are self-lubricating and hence no greasing is required.The apparatus (RB flask, still head, mercury pocket, receiver adapter, air condensor and receiver) is washed and dried as mentioned for the Kjeldahl’s flask (Page no. ). For drying the water condenser, the inner tube of the water condenser is washed and rinsed with acetone. The condenser is clamped vertically for sometime and then hot air is blown through the inner tube to dry it. The water condenser is generally not dried in the oven.All other glass apparatus is cleaned and dried like the kjeldahl flask

Note: The outer jacket of the water condenser is not dried.

Safe handling of mercury (a highly toxic element)Mercury (even when inhaled) is a highly toxic in nature. So mercury and mercury pocket should never be left uncovered while not in use.

AIM: Determination of the boiling point of the given organic compound by distillation method.

Learning objectives: After performing the experiment a student knows What is boiling point? Effect of pressure on the boiling point of a compound. Effect of volatile and non volatile impurities on the boiling point of

a compound Various methods and different types of apparatus used for boiling

point determination Significance of correct boiling point determination in the

characterization of an unknown organic compound Handling of mercury( a highly toxic element)

Requirements:ApparatusRound bottom flask, still head,

ChemicalsGiven sample (~ 20 mL), mercury.

collecting flask, mercury jacket, receiver adapter, thermometer, air condenser/ water condenser, sand bath, clamp and clamp stand and Bunsen burner.

Note: All the glass apparatus with I/C joints.

Procedure

1. Take a clean and dry distillation flask of 50 mL capacity.2. Clamp it on a stand about 12 inches from the base level.3. Transfer the liquid (about 20 mL), the boiling point of which has to be

determined) into a flask through a dry funnel.4. Put 3-4 pieces of pumice stones into the flask to prevent bumping and to

promote uniform boiling.5. Fix the still head on the round-bottomed flask containing the liquid

compound.6. Fix the mercury jacket containing liquid mercury (3-4 mL) on the still head.7. Place a thermometer in the mercury jacket such that the bulb of thermometer

is immersed in mercury.8. Attach a condenser (if the boiling point of the liquid is greater than 1500C,

an air condenser is used; if it is less than 1500C, a water condenser is used to condense the vapours and form liquid droplets), to the side tube of the still head.

9. Keep a dry receiver at the other end of the condenser for collecting the distillate.

10.Circulate water through the condenser (if a water condenser is used).11.Heat the flask slowly with a Bunsen burner so that the liquid distils slowly at

the rate of half a milliliter per minute.12.Note the constant temperature at which the liquid distils,( the temperature

rises rapidly until it is near the boiling point of the liquid, and then slowly and finally remains practically constant at the boiling point).

Fig. : Determination of boiling point by distillation method

DIAGRAM----CLAMPS ETC. MUST BE SHOWNNotes:1. The pumice stones should be added before starting heating and never while

heating.2. The mercury jacket should always be kept corked while not in use, as its

vapours are highly toxic.

3. The first few drops of the distillate should be discarded and the remaining distillate should be collected in a dry receiver.

4. A few drops of the liquid should be left undistilled in the round-bottomed flask at the end of the distillation process.

5.Boiling point of a liquid compound may be determined using the apparatus shown in fig. NO.Sources of errors:

1. Thermometer is not calibrated.2. The heating is not uniform.3. Delay in noting down the temperature at which the sample boils.

Precautions: 1. Wear safety glasses 2. Clamp the distillation and the receiving flasks securely but not too tight to

break the glassware.3. Handle mercury jacket and thermometer carefully.4. Note down the boiling point only when the temperature becomes constant.5. The assembly should be air-tight and there should be no leakage of vapours

of the liquid being distilled.6. The pumice stones should be added before starting heating and never while

heating.7. dry the distillation apparatus because in case the liquid to be distilled is

immiscible with water, a turbid and not a clear liquid is obtained even after distillation.

Result: The boiling point of the given organic compound is found to be ……….

II. Kjeldahl’s method (Siwoloboff’s method)

Requirements:ApparatusKjeldahl’s flask or a beaker, ignition tubes, a thin capillary tube, sand bath, clamp and clamp stand, thermometer fitted in the cork, dropper, rubber band, and Bunsen

ChemicalsGiven sample, concentrated sulphuric acid.

burner.

Procedure: 1. In Siwoloboff’s method, take a small quantity of the liquid(few drops) in an

ignition tube (6cmX0.3cm). 2. Place a sealed capillary tube with its open end in the liquid (the upper end

being sealed). This prevents superheating and provides a nucleus for bubbles to form.

3. Attach the ignition tube to the bulb of the thermometer with a rubber band. The rubber band should be above the level of bath liquid.

4. Lower the thermometer with the ignition tube in the concentrated sulphuric acid (or liquid paraffin) kept in a Kjeldahl’s flask or a beaker, taking care to keep the bulb of the thermometer and the lower end of the ignition tube at the same level.

5. Heat the Kjeldahl’s flask or the beaker gradually with a small non-luminous Bunsen flame, rotating to ensure even heating; there will be a slow escape of bubbles from the end of the capillary tube.

6. When the temperature is near the expected boiling point, decrease the rate of heating so that the temperature rises at about the rate of 1-2ºC per minute.

7. Note the temperature at which a rapid and continuous stream of bubbles is emitted from the capillary tube. This temperature is taken as the boiling point of the liquid.

8. When the source of heat is removed, the speed at which the bubbles are given off will slacken.

9. At the boiling point, the last bubble makes its appearance and tends to be sucked back into the tube.

Note: The capillary tube should be very thin walled, about 8cm long and 1mm in diameter.

Sources of errors:1. Thermometer is not calibrated.2. The sample is impure.3. The heating is not uniform.4. Delay in noting down the temperature at which the sample boils.

Precautions:

1. Wear safety glasses.2. Capillary tube breaks easily, hence handle it carefully.3. Seal the capillary tube from one end.4. Place a sand bath below the flask while detrming the boiling point so that if

the flask breaks the person performing the experiment is safe.5. Clamp the Kjeldahl’s flask properly, neither too tight nor too loose.6. Handle thermometer carefully.7. The bulb of the thermometer and the sample in the ignition tube should be

well immersed in the bath liquid.8. Use a grooved cork with thermometer.9. Rotate the burner throughout so that heating is uniform.10.Ensure that the wall of the ignition tube touches the bulb of the thermometer.11.Note down the boiling point only when a continuous stream of bubbles is

obtained. 12.If the acid in the concentrated sulphuric acid bath becomes coloured through

contact with organic matter, it can be easily decolourized by adding a crystal of potassium nitrate and heating.

13.After determination of the boiling point the acid bath should be allowed to cool to room temperature.

Fig. : Siwoloboff’s method for determination of boiling point

Questions for viva-voce:

1. What is boiling point?2. List out the possibilities of having sharp boiling point.3. Why is it necessary to use grooved cork to fit the thermometer in the

Kjeldahl’s flask?4. Why do you use pumice stones during distillation?5. Why should the pumice stones be added before stating heating and not while

heating? 6. Explain the criterion of purity in case of a liquid organic compound.7. What do you mean by the literature boiling point of a compound?8. Why is it advised to slow down heating near the boiling point of the

compound?9. Why is it necessary to keep a sand bath below the Kjeldahl’s flask while

determining the b. pt.?10.Describe Siwoloboff’s method of boiling point determination.11.Describe distillation method of boiling point determination.12.What are the advantages of distillation method over Kjeldahl’s method?13.What do you mean by the calibration of the thermometer?

Chapter

CHROMATOGRAPHY

Introduction

Chromatography is a separation technique widely used in laboratories. It can be

used for separation of solids, liquids and gases and hence is more useful than other

common techniques like crystallization (which is used for purification of solids

only), distillation ( used for purification of liquids only) etc. which have limited

applications. Other advantage of chromatography as a technique is that it can be

used for separation and purification on a micro as well as macro scale and unlike

crystallization and distillation, the separation procedure is carried out under mild

conditions.

The technique was first used in 1903 by Tswett for the separation of coloured

substances, hence the name chromatography. Now the technique is used for

separation of colourless compounds as well.

Theory

The basic principle of chromatography is that a substance to be purified is

partitioned or distributed between two different phases. One which is fixed is

called the stationary phase and other which moves is called the mobile phase.

The substance to be purified is applied on the stationary phase and is then made to

move on the stationary phase along with the mobile phase.

Different substances travel different lengths from the point of application with the

moving phase. In partition chromatography (discussed below) the distance

travelled depends on the partition coefficient (K) of the substance and is

K= Conc. of substance in solvent A/ Conc. of substance in solvent B

Different compounds have different partition coefficients and thus the components

of the mixture get separated.

The distance travelled by a substance from the point of application on the

chromatogram is given by the Rf value (Retention factor)

Rf= Distance moved by the substance/ Distance moved by the moving phase or

solvent

The Rf value depends on the following

1. Nature of the substance.

2. Nature of the Stationary phase

3. Nature of the moving phase

When the compounds are coloured, coloured spots are observed on the

chromatogram at different heights from the point of application.

If the compounds to be purified and impurities are colourless, these are visualized

by (a) application of chemicals called visualizing agents which chemically

combine with colourless compounds and produce coloured spots or (b) viewing the

spots under lights of different wave lengths for example U.V. light.

Chromatography

Adsorption (Solid stationary phase) Partition (liquid stationary phase)

Liquid mobile phase Gaseous mobile phaseLiquid mobile phase Gaseous mobile phase

Chromatographic techniques are broadly classified on the basis of the physical

state of the stationary and the moving phase. It is called adsorption

chromatography when a solid is the stationary phase and partition chromatography

when a liquid is stationary phase.

Thus depending on the nature of stationary phase and mobile phase, four types of

combinations are possible

i. Liquid-solid

ii. Gas-solid

iii. Liquid-liquid

iv. Gas-liquid

Following are the common types of chromatographic techniques

a)Paper chromatography (liquid-liquid) i.e. the water held on the filter paper

by adsorption on cellulose molecules in the stationary phase and a liquid phase

is used as the mobile phase

b)Thin layer chromatography (solid-liquid): A solid supported on a plate is

the stationary phase and a liquid is the mobile phase. The solid stationary phase is

adhered onto a glass or a metal plate as a thin film ( and hence the name thin layer

chromatography)

c)Column chromatography (solid-liquid or liquid-liquid): The solid stationary

phase or the liquid stationary phase supported on a solid is placed in a tube

called column and a liquid is the mobile phase

d)Gas chromatography: Stationary phase is solid and mobile phase is a gas

e)HPLC: High Pressure Liquid Chromatography

The choice of chromatography technique for the purification of an organic

compound or the selection of stationary or mobile phase is generally empirical as

there is no way of predicting the best system, though a lot of information can be

obtained from literature. It is best to try simple techniques like paper or thin layer

chromatography to standardize the system that will work for a given compound or

a mixture of compounds and then use more sophisticated techniques for separation.

Following is the list of rough guide that may be considered

Covalent compounds of similar

chemical nature

Partition chromatography

Covalent compounds of different

chemical nature

Adsorption

Volatile compounds or gaseous

compounds

Gas chromatography

Ions or ionic compounds Paper, TLC, column, ion exchange

etc.

Ionic compounds from covalent

compounds

Ion exchange

Paper chromatography

It is a typical example of partition chromatography where stationary and mobile

phase are both liquids.

The stationary phase as already mentioned is water, adsorbed on the cellulose

molecules of the Whatman filter paper, which is used for application of the

compound (see section ?). The Whatman filter paper thus acts as a support for

water, the stationary phase.

The moving phase is a solvent or a mixture of solvents, the polarity of which is

decided on the basis of how strongly the compound to be purified is adsorbed on

the stationary phase.

Paper chromatography may further be classified on the basis of the direction of the

flow of the moving phase (solvent).

i. Ascending chromatography: Where the solvent (moving phase) is placed in

a jar and when one end of the paper strip containing the spotted compound is

dipped in the solvent, it rises up or ascends.

ii. Descending chromatography: The solvent (moving phase) descends down

the strip.

iii. Horizontal chromatography: The solvent moves horizontally along the paper

General Procedure

A drop of the solution of compound to be purified is placed on a marked position

on the Whatman filter paper. When the spot has dried, the paper is placed in a

suitable closed apparatus containing the liquid mobile phase.

The solvent percolates through the fibres of the paper by capillary action and

moves the components of the mixture to different extents in the direction of the

flow. Different compounds move to different levels with the solvent.

The distance travelled by the substance is noted as its Rf (Retention factor)

Thin Layer Chromatography

In this technique the stationary phase is solid and the mobile phase is a liquid. The

stationary phase (which is generally silica or alumina) is fixed on the surface of a

glass plate (different sizes are in use) or a thin metal sheet ( e.g. aluminium ) with

the help of a binding agent such as Calcium sulphate

The surface of silica or alumina may be modified according to the requirement i.e.

made acidic, basic, neutral, coated with fluorescent chemicals etc.

The experimental technique (section ? ) remains similar to the paper

chromatography

The Paper or Thin Layer chromatographic techniques can be used

a) For identification of the number of components in a mixture of compounds

b) Purification of a compound

c) Establishing the identity of the two compounds ( comparing their Rf value

under identical experimental conditions)

d) Monitoring the progress of a reaction etc.

Column chromatography

A glass tube (with a nozzle at one end and open at the other end) called a column is

used to hold the stationary phase. The stationary phase may be a solid, which is

packed in the column (adsorption chromatography) or a liquid supported on a solid

(partition chromatography).

The mobile phase which is generally a liquid moves through the column (packed

with stationary phase) and carries the components of the mixture along with it,

which move with different speeds in the column and thus get separated.

The mobile phase or the liquid is called the eluent and the term elution means

allowing a solvent or a mixture of the solvents to run through the column until the

separation or purification of the compounds is achieved.

The separated constituents of the mixture can be seen while pacing down the

column, if they are coloured, otherwise the eluate is collected in different fractions,

the liquid distilled from each fraction separately and the residue is checked ( by

paper or thin layer chromatography) for its purity.

Sometimes a transparent nylon tube is used as column, when the components of

the mixture have separated in the tube, the portion containing each component is

cut, the stationary phase separated out and the solute is extracted using a suitable

solvent, the solid phase is filtered and distillation of the solvent gives the residue

i.e. the required compound.

Column chromatography can be used for purification of a given compound or for

the separation of a mixture of compounds.

PROCEDURE

(a) Ascending Paper Chromatography

1) Place a suitable solvent ( ~ 10 -15 ml ) in a chromatography jar fitted

with a cork and a hook ( fig. ? ). Cover the jar with the lid and allow

it to get saturated with the solvent vapours

2) Take a strip ( figure ?) of Whatman no 1 filter paper

3) Draw a line (using a pencil) about 2.5 cm from one edge of the filter

paper

4) Using a fine capillary, apply the solution of the sample on the line

( one spot of the sample solution for finding out the number of

components in a given mixture of compounds, two or more for

comparison of the components of the mixture with pure compounds)

Repeat the spotting two three times at the same point and dry after

each application.

5) Hook the chromatogram with the lid and lower the spotted end into

the jar containing the solvent such that (i) the paper does not touch

the inner sides of the jar (ii) the lower end of the paper but not the

spots dip into the solvent (figure ?)

6) Cover the jar and allow the set up to stand undisturbed.

7) When the solvent reaches near the upper end of the paper, take it out ,

mark the solvent front and allow the paper to dry.

8) If the components are coloured, spots will be visible (the no of spots

corresponds to the no of components in the mixture). Otherwise spray

the paper with the visualizing agent or view the chromatogram using a

U.V lamp.

9) Mark the spots, measure the distance travelled by the solvent and each

component of the mixture and calculate the Rf value of each.

DIAGRAMS

Circular Chromatography

1) Take a clean dry petri dish, place ~ 10-15mL of the solvent in it and

cover it with a lid. Allow the dish to get saturated with the solvent

vapors’.

2) Take a circular Whatman filter paper, slightly bigger in diameter than

the dish. Apply the given solution in the centre of the paper as

described in ascending chromatography.

3) Make a small hole in its centre through the spot and pass a cotton

wick through it (figure ?)

4) Dip the wick into the solvent in the petri dish such that the

chromatogram rests on the rim of the dish.

5) Cover the petri dish and allow the solvent move horizontally along the

paper.

6) When the solvent reaches near the outer edge of the paper, take it out,

mark the solvent front and dry the paper.

7) Coloured rings are observed if the components are coloured.

8) Spray with locating agent, if no spots are visible

9) Calculate the Rf value.

DIAGRAMS

Descending Chromatography

1) Place the solvent in the boat at the top of the jar and cover it

2) Apply the sample on the chromatogram as described in ascending

chromatographic technique

3) Place the chromatogram in the solvent taking all care suggested above.

4) Rest of the procedure is same as explained above.

DIAGRAMS

Thin Layer Chromatography

1) Take a chromatography jar, add 10-15 mL of the required solvent and cover

it with a lid.

2) Take a glass plate coated with the stationary phase.

3) Apply the solution of the compound ~2 cm. above the edge of the plate as

explained in ascending chromatography

4) Place the plate in the jar

5) Follow the procedure under ascending paper chromatography

DIAGRAMS

Precautions

1) Use a fine capillary tube for applying the solution.

2) Apply the sample carefully at the same point, dry the spot every time.

3) Saturate the air in the jar with the solvent vapours.

4) Place the chromatogram carefully in the jar such that it does not touch

the sides of the jar,the spotted portion is not dipped in the solvent and

the paper is straight.

5) Mark the solvent front immediately after taking out the chromatogram.

Column Chromatography

1) Take a glass column with a nozzle at one end

2) Select the size of column depending on the amount of the substance to be

purified.

3) Insert a small plug of cotton through the open end of the column and using a

glass rod, push it near the nozzle.

4) Clamp the column vertically on a stand.

5) Take the required amount of silica gel (or any other solid to be used as

stationary phase) in a dry beaker, add the solvent and stir to obtain a slurry.

6) Place a funnel on the open end of the column, add the slurry through it into

the column (continuously stir the slurry with a glass rod).

7) Open the nozzle of the column while adding the slurry and collect the

solvent while it drops.

8) Add the slurry till at least two third of the column is packed with the

stationary phase

9) Take the solution of the compound and pour it into the column through the

funnel .The solution of the compound will slowly pass into the stationary

phase.

10) Add the eluting solvent from the top and keep collecting the liquid

that drops from the nozzle

11) As the components of the mixture descend down, coloured bands are

seen at different levels in the column.

12) Collect the fraction containing the compound in a separate conical

flask.

13) Transfer the liquid into the distillation flask and remove the solvent by

distillation. The residue contains the purified compound.

14) Similarly elute other components of the mixture from the column.

Precautions

1) Use a clean and dry column

2) While adding the slurry of the stationary phase, continuously stir it to obtain

a uniform packing in the column. Otherwise the separation of components of

the mixture is not clean.

3) Do not allow the column to dry during the experiment. Always keep the

column immersed in the solvent.

4) Do not run the solvent rapidly through the column. Allow it to pass through

the column slowly to get a clean separation of the components.

Questions for viva- voce

1) What are the advantages of chromatographic technique over other separation

techniques

2) Why it was called chromatography?

3) How is the method used for separation of colourless compounds?

4) What do understand by the term stationary and mobile phase? Give a few

examples.

5) What is the difference between adsorption and partition chromatography?

6) What is the difference between ascending, descending and circular

chromatography?

7) Name the stationary phase in paper chromatography.

8) What is TLC?

9) Name a few compounds used as stationary phase in TLC.

10) What do you understand by the term visualising or spraying agent?

Give a few examples.

11) What is the visualising agent for amino acids? Discuss the chemistry

involved.

12) What is column chromatography?

13) What is Rf?

Experiment

Aim – To separate the given mixture of amino acids by ascending paper

chromatography and calculate the Rf of each component.

Learning objectives

A students learns

1) The meaning and importance of chromatography as a method of separation.

2) Terms – stationary and moving phase,, visualising and spraying agent, Retention

factor (Rf)

3) And understands the concept of distribution and partition.

4) How to separate a mixture of organic compounds.

5) Understands the chemistry of reaction of an amino acid with

ninhydrin( used as the visualizing agent)

Requirements

Apparatus Chemicals

Chromatography jar fitted with a cork

and a hook

Whatman filter paper strip( )

Capillary tube

Sprayer

Solvent- n- Butanol: acetic acid:water

(4:1:5)

Given solution of amino acids*

Spraying or visualizing agent: 1%

Ninhydrin solution

REACTION OF NINHYDRIN WITH AMINO ACIDS----IS TO BE GIVEN

HERE

Note

*A mixture of any of following: serine & isoleucine, glycine & aspartic acid,

alanine & leucine etc. can be used for best separation of the components of

mixture as their Rf values are different in the given solvent system.

1) The number of components in a given mixture of amino acids can also be

identified

a) by circular or descending paper chromatography.

b) By TLC

2) Identity or non identity of two samples can also be established by

By spotting the samples on the same chromatogram and compairing their Rf

value

Aim –To indentify the number of carbohydrates present in the given mixture

Requirements

Apparatus Chemicals

Same as in the above experiment Solvent – ( Butanol: Acetone: Water;

4:5:1) or (Methyl Ethyl Ketone: Acetic

Acid: Water; 3:1:1) or ( Butanol:

Acetic Acid: Water; 4:1:5)

Solution of carbohydrates**

Spraying or visualizing agent:

(1)Aniline Hydrogen

Phthalate (Spray & heat

for 4-5 minutes at 100

degree centigrade)

(2)Anisidine Phthalate

**: Any one of the following combination may be given for chromatography:

(Glucose and Fructose), (Glucose and Sucrose), (Fructose and Sucrose)

PURIFICATION OF THE ORGANIC COMPOUNDS CRYSTALLISATION

Introduction

An organic compound which is either synthesized in the laboratory or isolated from any natural source is rarely obtained in a pure form. It is invariably contaminated with other compounds i.e. impurities. Purification of the compounds thus becomes very important, for its identification as the pure compound has a sharp and well-defined melting point and for its final use as a pharmaceutical, a food colorant, flavour, perfume, pesticide, fertilizer etc.

A number of methods have been used for the purification of the compounds. For example, a solid compound may be purified by one or a combination of the follwing methods:

Crystallization Fractional Crystallization Sublimation Solvent extraction Chromatography etc.

Theory

Purification by crystallizationThe principle underlying crystallization is that the solubility of a compound in a solvent increases on increasing the temperature and vice-versa. So when a saturated solution of a compound is prepared in a suitable solvent at high temperature (i.e. boiling point of the solvent), the amount of solute dissolved is much more as compared to what would have dissolved at room temperature or a lower temperature, so when a hot solution is allowed to cool, the extra or excess

solute that is dissolved separates or crystallizes out and can be obtained by filtration.

This technique is used as a simple method for purification of compounds. The impure compound is dissolved in a solvent such that the impurities are either insoluble even in the hot solvent or so soluble that these remain dissolved even at a lower temperature.

Hence for purification of a solid compound by crystallization, the choice of the solvent is very important.

The various steps involved in the crystallization of a solid compound:

Selection of a solvent:In order to select a solvent for purification of the compound, take about 50mg of the compound in a test tube ,add to it about 2-3ml of the solvent (water, methanol, ethanol, ethyl acetate etc. are some common organic solvents used for crystallization) and heat the test tube in a hot water bath containing inflammable liquids or directly on a flame if water is used as asolvent. To choose a suitable solvent, the following points should be kept in mind:

6. The solvent should be inert, that is, it should not react with the compound to be purified.

7. The compound to be purified should be highly soluble in the solvent at high temperatures and almost insoluble at room temperatures.

8. The impurities should be either soluble in the solvent at room temperature such that they remain in the mother liquor and do not crystallize out or are insoluble even at high temperatures and thus can be removed by hot filtration.

9. The boiling point of the solvent should be preferably lower than the melting point of the compound.

10.The rule of ‘like dissolves like’ should be kept in mind while selecting a solvent.

Note:If a single solvent does not meet the above criteria, a mixture of solvents (which are miscible with each other e.g water-ethanol, petroleum ether-ethyl acetate, etc.) may be used for crystallization.

Dissolution of the solute in the required amount of solvent: Take the impure solid in a clean test tube or a boiling tube. Add a few milliliters (4-5) of the solvent and heat the solution directly on a flame with water as solvent or in case an inflammable organic solvent is being used as solvent, heat the solution on a water bath with constant stirring using a glass rod. If the solid does not dissolve, add a few milliliters of the solvent and heat again (do not add excess of solvent). If near the end of the dissolution process, it appears that an additional amount of the solvent is not dissolving any more of the solid, then this small amount of undissolved solid is likely to be an insoluble impurity. Do not add any more solvent but filter the hot solution through a fluted filter paper (Fig. 1).

Fig. 1: How to fold to get a fluted filter paper.

Note:If a mixture of two solvents is used for crystallization-dissolve the solute in a minimum amount of the solvent in which it is more soluble and then add the other solvent (in which the compound is insoluble or less soluble) dropwise till turbidity just appears. Heat the turbid solution directly on a flame or a hot water bath (if inflammable solvents are being used) to obtain a clear solution and keep aside for crystallization.

Decolourization:If the solution obtained is highly coloured, add a small amount of animal charcoal, boil the solution for some time. (The coloured impurities are adsorbed on the surface of charcoal.) Filter the hot solution through a fluted filter paper and allow the filtrate to cool for the separation of crystals.

Crystallization:

Do not cool the solution obtained above rapidly, allow it to slowly cool to room temperature. If no crystals are obtained, cool the solution in an ice-bath or scratch the sides of the test tube with a glass rod, just near the surface of the solution to initiate crystallization or add 2-3 crystals of the solid crude compound to induce crystal formation.

Filtration and drying:Filter the crystals by vacuum filtration and collect the mother liquor in a clean dry boiling tube. Cool the mother liquor in an ice bath, concentrate and collect to obtain the second crop of crystals. Filter and air dry or keep in a vacuum desiccator.

Some important terms involved are:Crystallization point:The stage at which solution becomes saturated with respect to the solute is called crystallization point.

Mother liquor:The residual fluid filtrate left behind after the separation of crystals from the saturated solution is called mother liquor.

Bumping:It is the spurting out of the solution while being heated. To some extent, bumping can be reduced by adding two or three small pieces of pumice stone to the solution being heated.

Seeding:Sometimes, no crystals are obtained on cooling the solution even though it is concentrated enough. Then during the cooling and scratching, a minute quantity of the crude material may be added to “seed” the solution. It is called “seeding” and it facilitates initial crystallization.

Aim: Purification of the given compound by crystallization using:(i) water(ii) alcohol as the solvent.

Learning Objectives: After performing the experiment, a student:

(ix) learns the importance of purification of the compound.

(x) can choose an appropriate solvent for crystallization of unknown compound.

(xi) learns that on the basis of difference in solubilities, the impurities can be removed to obtain pure compound.

(xii) learns to handle and heat inflammable organic solvents.(xiii) learns to make a saturated solution.(xiv) learns to make a fluted filter paper.(xv) learns the use of vacuum pump.(xvi)learns about various filtration apparatus used.

Requirements:

ApparatusTest tubes, boiling tubes, funnel, filtering tube, filter button, Buchner funnel, Buchner flask, tongs, glass rod, etc.

ChemicalsGiven solid compound, alcohol and charcoal.

Procedure

Crystallization using water as solvent:5. Take ~0.5g of the impure compound in a boiling tube and add minimum

amount of water (about 5mL in the beginning).6. Heat the contents to boiling by holding the boiling tube with a test tube

holder (stir while heating using a glass rod). Add more hot water if required. 7. Remove the suspended impurities by filtration of the hot solution. To carry

out hot filtration, filter the boiling hot solution through a fluted filter paper in a pre-heated funnel. Collect the filtrate in a pre-heated boiling tube (carry out the pre-heating by heating the boiling tube, funnel and fluted filter paper in an oven).

8. Allow the filtrate to cool slowly in air to room temperature.

Crystallization using alcohol as solvent:3. Take 0.5g of the impure compound in a boiling tube and add minimum

amount of alcohol (about 5mL in the beginning). 4. Heat the contents to the boiling point of the alcohol in a boiling water bath.

Add more hot alcohol, if required, to dissolve the compound. Then follow step 3 and 4 as above.

Filtration: Using a filtering tube and a funnel(xv) Take a rubber cork to fit into the filtering tube.(xvi) Make a hole in the centre of the cork and fit a filter funnel into it, the

fitting should be air-tight.(xvii) Fix the cork with the funnel in the filtering tube.(xviii)Clamp it near the filtration pump.(xix) Fix a filter button or a small filter disc to cover the stem of the funnel.(xx) Cut a round piece of filter paper (slightly bigger than the button or disc).(xxi) Wet the filter paper with water. Place it on the button or filter disc.(xxii) Connect the side tube of the filtering tube to the vacuum pump.(xxiii)Switch on the vacuum pump.(xxiv) Transfer the crystals along with mother liquor on the funnel.(xxv) Allow the mother liquor to drain completely.(xxvi) Disconnect the connecting tube from the pump.(xxvii) Transfer the crystals from funnel to a filter paper. (xxviii) Allow to dry in air.

Note:A Buchner funnel and a Buchner flask may be used instead of a funnel and a filtering tube for filtration if the amount of the solid is more.

Filtering tube Buchner flask and funnel

Vacuum pump

Fig. : Vacuum filtration assembly

Precautions8. Do not add excess of the solvent for the dissolution of the solute.9. While dissolving the impure sample in an inflammable solvent like alcohol,

do not heat the test tube directly on a flame but heat it in a hot water bath. (Either clamp it or hold with a test tube holder)

10.Add a few pieces of pumice stones before heating to avoid bumping. 11.Do not take alcohol near the flame. 12.Allow the solution to cool slowly for crystallization and do not disturb it

during the crystallization process.13.Spread the crystals on the filter paper for drying.14.Do not crush the crystals while drying.

ResultTotal yield of the crystallized organic sample obtained =

Note: Prepare a hot saturated solution of the compound for crystallization. Add pumice stones before heating the solution and never while heating. In case of purification of a compound which is highly insoluble in the

solvent at room temperature, a slight excess of the solvent is required. But in other cases especially while using alcohol as solvent, one must use minimum amount of the solvent.

In case no crystals appear even after keeping the filtrate at room temperature, it may be due to the fact that filtrate is not saturated. So concentrate it to reduce the volume by heating it directly over flame in case of water as solvent and by heating it in water bath in case of alcohol as solvent. Scratch the sides of the test tube with a glass rod, just near the surface of the solution to initiate crystallization.

To ensure the purity of crystallized sample, determine its melting point. In general it may be said that a pure compound has usually a sharp melting point i.e. the substance melts entirely within a range of about 1ºC, whereas an impure substance does not have a sharp melting point and will therefore melt slowly and indecisively over a range of several degrees.

Questions for viva-voce:10.Describe the principle behind crystallization.11.Discuss the various steps involved in crystallization.12.What is fluted filter paper and how is it made?13.How to select a suitable solvent for crystallization of an organic compound?14.Why is it necessary to use pumice stone while heating the solution?15.How are the soluble coloured impurities removed?16.What is the advantage of vacuum filtration over normal filtration using

funnel and filter paper?17.What is ‘mother liquor’?

18.Name some solvents which can be used for crystallization.

Chapter

CHROMATOGRAPHY

Introduction

Chromatography is a separation technique widely used in laboratories. It can be

used for separation of solids, liquids and gases and hence is more useful than other

common techniques like crystallization (which is used for purification of solids

only), distillation ( used for purification of liquids only) etc. which have limited

applications. Other advantage of chromatography as a technique is that it can be

used for separation and purification on a micro as well as macro scale and unlike

crystallization and distillation, the separation procedure is carried out under mild

conditions.

The technique was first used in 1903 by Tswett for the separation of coloured

substances, hence the name chromatography. Now the technique is used for

separation of colourless compounds as well.

Theory

The basic principle of chromatography is that a substance to be purified is

partitioned or distributed between two different phases. One which is fixed is

called the stationary phase and other which moves is called the mobile phase.

The substance to be purified is applied on the stationary phase and is then made to

move on the stationary phase along with the mobile phase.

Different substances travel different lengths from the point of application with the

moving phase. In partition chromatography (discussed below) the distance

travelled depends on the partition coefficient (K) of the substance and is

K= Conc. of substance in solvent A/ Conc. of substance in solvent B

Different compounds have different partition coefficients and thus the components

of the mixture get separated.

Chromatography

Adsorption (Solid stationary phase)

Liquid mobile phase Gaseous mobile phase

Partition (liquid stationary phase)

Liquid mobile phase Gaseous mobile phase

The distance travelled by a substance from the point of application on the

chromatogram is given by the Rf value (Retention factor)

Rf= Distance moved by the substance/ Distance moved by the moving phase or

solvent

The Rf value depends on the following

4. Nature of the substance.

5. Nature of the Stationary phase

6. Nature of the moving phase

When the compounds are coloured, coloured spots are observed on the

chromatogram at different heights from the point of application.

If the compounds to be purified and impurities are colourless, these are visualized

by (a) application of chemicals called visualizing agents which chemically

combine with colourless compounds and produce coloured spots or (b) viewing the

spots under lights of different wave lengths for example U.V. light.

Chromatographic techniques are broadly classified on the basis of the physical

state of the stationary and the moving phase. It is called adsorption

chromatography when a solid is the stationary phase and partition chromatography

when a liquid is stationary phase.

Thus depending on the nature of stationary phase and mobile phase, four types of

combinations are possible

v. Liquid-solid

vi. Gas-solid

vii. Liquid-liquid

viii. Gas-liquid

Following are the common types of chromatographic techniques

a)Paper chromatography (liquid-liquid) i.e. the water held on the filter paper

by adsorption on cellulose molecules in the stationary phase and a liquid phase

is used as the mobile phase

b)Thin layer chromatography (solid-liquid): A solid supported on a plate is

the stationary phase and a liquid is the mobile phase. The solid stationary phase is

adhered onto a glass or a metal plate as a thin film ( and hence the name thin layer

chromatography)

c)Column chromatography (solid-liquid or liquid-liquid): The solid stationary

phase or the liquid stationary phase supported on a solid is placed in a tube

called column and a liquid is the mobile phase

d)Gas chromatography: Stationary phase is solid and mobile phase is a gas

e)HPLC: High Pressure Liquid Chromatography

The choice of chromatography technique for the purification of an organic

compound or the selection of stationary or mobile phase is generally empirical as

there is no way of predicting the best system, though a lot of information can be

obtained from literature. It is best to try simple techniques like paper or thin layer

chromatography to standardize the system that will work for a given compound or

a mixture of compounds and then use more sophisticated techniques for separation.

Following is the list of rough guide that may be considered

Covalent compounds of similar

chemical nature

Partition chromatography

Covalent compounds of different

chemical nature

Adsorption

Volatile compounds or gaseous

compounds

Gas chromatography

Ions or ionic compounds Paper, TLC, column, ion exchange

etc.

Ionic compounds from covalent

compounds

Ion exchange

Paper chromatography

It is a typical example of partition chromatography where stationary and mobile

phase are both liquids.

The stationary phase as already mentioned is water, adsorbed on the cellulose

molecules of the Whatman filter paper, which is used for application of the

compound (see section ?). The Whatman filter paper thus acts as a support for

water, the stationary phase.

The moving phase is a solvent or a mixture of solvents, the polarity of which is

decided on the basis of how strongly the compound to be purified is adsorbed on

the stationary phase.

Paper chromatography may further be classified on the basis of the direction of the

flow of the moving phase (solvent).

iv. Ascending chromatography: Where the solvent (moving phase) is placed in

a jar and when one end of the paper strip containing the spotted compound is

dipped in the solvent, it rises up or ascends.

v. Descending chromatography: The solvent (moving phase) descends down

the strip.

vi. Horizontal chromatography: The solvent moves horizontally along the paper

General Procedure

A drop of the solution of compound to be purified is placed on a marked position

on the Whatman filter paper. When the spot has dried, the paper is placed in a

suitable closed apparatus containing the liquid mobile phase.

The solvent percolates through the fibres of the paper by capillary action and

moves the components of the mixture to different extents in the direction of the

flow. Different compounds move to different levels with the solvent.

The distance travelled by the substance is noted as its Rf (Retention factor)

Thin Layer Chromatography

In this technique the stationary phase is solid and the mobile phase is a liquid. The

stationary phase (which is generally silica or alumina) is fixed on the surface of a

glass plate (different sizes are in use) or a thin metal sheet ( e.g. aluminium ) with

the help of a binding agent such as Calcium sulphate

The surface of silica or alumina may be modified according to the requirement i.e.

made acidic, basic, neutral, coated with fluorescent chemicals etc.

The experimental technique (section ? ) remains similar to the paper

chromatography

The Paper or Thin Layer chromatographic techniques can be used

e) For identification of the number of components in a mixture of compounds

f) Purification of a compound

g) Establishing the identity of the two compounds ( comparing their Rf value

under identical experimental conditions)

h) Monitoring the progress of a reaction etc.

Column chromatography

A glass tube (with a nozzle at one end and open at the other end) called a column is

used to hold the stationary phase. The stationary phase may be a solid, which is

packed in the column (adsorption chromatography) or a liquid supported on a solid

(partition chromatography).

The mobile phase which is generally a liquid moves through the column (packed

with stationary phase) and carries the components of the mixture along with it,

which move with different speeds in the column and thus get separated.

The mobile phase or the liquid is called the eluent and the term elution means

allowing a solvent or a mixture of the solvents to run through the column until the

separation or purification of the compounds is achieved.

The separated constituents of the mixture can be seen while pacing down the

column, if they are coloured, otherwise the eluate is collected in different fractions,

the liquid distilled from each fraction separately and the residue is checked ( by

paper or thin layer chromatography) for its purity.

Sometimes a transparent nylon tube is used as column, when the components of

the mixture have separated in the tube, the portion containing each component is

cut, the stationary phase separated out and the solute is extracted using a suitable

solvent, the solid phase is filtered and distillation of the solvent gives the residue

i.e. the required compound.

Column chromatography can be used for purification of a given compound or for

the separation of a mixture of compounds.

PROCEDURE

(a) Ascending Paper Chromatography

10) Place a suitable solvent ( ~ 10 -15 ml ) in a chromatography jar

fitted with a cork and a hook ( fig. ? ). Cover the jar with the lid and

allow it to get saturated with the solvent vapours

11) Take a strip ( figure ?) of Whatman no 1 filter paper

12) Draw a line (using a pencil) about 2.5 cm from one edge of the

filter paper

13) Using a fine capillary, apply the solution of the sample on the

line ( one spot of the sample solution for finding out the number of

components in a given mixture of compounds, two or more for

comparison of the components of the mixture with pure compounds)

Repeat the spotting two three times at the same point and dry after

each application.

14) Hook the chromatogram with the lid and lower the spotted end

into the jar containing the solvent such that (i) the paper does not

touch the inner sides of the jar (ii) the lower end of the paper but not

the spots dip into the solvent (figure ?)

15) Cover the jar and allow the set up to stand undisturbed.

16) When the solvent reaches near the upper end of the paper, take

it out , mark the solvent front and allow the paper to dry.

17) If the components are coloured, spots will be visible (the no of

spots corresponds to the no of components in the mixture). Otherwise

spray the paper with the visualizing agent or view the chromatogram

using a U.V lamp.

18) Mark the spots, measure the distance travelled by the solvent

and each component of the mixture and calculate the Rf value of each.

DIAGRAMS

Circular Chromatography

10) Take a clean dry petri dish, place ~ 10-15mL of the solvent in it

and cover it with a lid. Allow the dish to get saturated with the solvent

vapors’.

11) Take a circular Whatman filter paper, slightly bigger in

diameter than the dish. Apply the given solution in the centre of the

paper as described in ascending chromatography.

12) Make a small hole in its centre through the spot and pass a

cotton wick through it (figure ?)

13) Dip the wick into the solvent in the petri dish such that the

chromatogram rests on the rim of the dish.

14) Cover the petri dish and allow the solvent move horizontally

along the paper.

15) When the solvent reaches near the outer edge of the paper, take

it out, mark the solvent front and dry the paper.

16) Coloured rings are observed if the components are coloured.

17) Spray with locating agent, if no spots are visible

18) Calculate the Rf value.

DIAGRAMS

Descending Chromatography

5) Place the solvent in the boat at the top of the jar and cover it

6) Apply the sample on the chromatogram as described in ascending

chromatographic technique

7) Place the chromatogram in the solvent taking all care suggested above.

8) Rest of the procedure is same as explained above.

DIAGRAMS

Thin Layer Chromatography

3) Take a chromatography jar, add 10-15 mL of the required solvent and cover

it with a lid.

4) Take a glass plate coated with the stationary phase.

6) Apply the solution of the compound ~2 cm. above the edge of the plate as

explained in ascending chromatography

7) Place the plate in the jar

8) Follow the procedure under ascending paper chromatography

DIAGRAMS

Precautions

6) Use a fine capillary tube for applying the solution.

7) Apply the sample carefully at the same point, dry the spot every time.

8) Saturate the air in the jar with the solvent vapours.

9) Place the chromatogram carefully in the jar such that it does not touch

the sides of the jar,the spotted portion is not dipped in the solvent and

the paper is straight.

10) Mark the solvent front immediately after taking out the

chromatogram.

Column Chromatography

15) Take a glass column with a nozzle at one end

16) Select the size of column depending on the amount of the substance to

be purified.

17) Insert a small plug of cotton through the open end of the column and

using a glass rod, push it near the nozzle.

18) Clamp the column vertically on a stand.

19) Take the required amount of silica gel (or any other solid to be used as

stationary phase) in a dry beaker, add the solvent and stir to obtain a slurry.

20) Place a funnel on the open end of the column, add the slurry through it

into the column (continuously stir the slurry with a glass rod).

21) Open the nozzle of the column while adding the slurry and collect the

solvent while it drops.

22) Add the slurry till at least two third of the column is packed with the

stationary phase

23) Take the solution of the compound and pour it into the column

through the funnel .The solution of the compound will slowly pass into the

stationary phase.

24) Add the eluting solvent from the top and keep collecting the liquid

that drops from the nozzle

25) As the components of the mixture descend down, coloured bands are

seen at different levels in the column.

26) Collect the fraction containing the compound in a separate conical

flask.

27) Transfer the liquid into the distillation flask and remove the solvent by

distillation. The residue contains the purified compound.

28) Similarly elute other components of the mixture from the column.

Precautions

5) Use a clean and dry column

6) While adding the slurry of the stationary phase, continuously stir it to obtain

a uniform packing in the column. Otherwise the separation of components of

the mixture is not clean.

7) Do not allow the column to dry during the experiment. Always keep the

column immersed in the solvent.

8) Do not run the solvent rapidly through the column. Allow it to pass through

the column slowly to get a clean separation of the components.

Questions for viva- voce

14) What are the advantages of chromatographic technique over other

separation techniques

15) Why it was called chromatography?

16) How is the method used for separation of colourless compounds?

17) What do understand by the term stationary and mobile phase? Give a

few examples.

18) What is the difference between adsorption and partition

chromatography?

19) What is the difference between ascending, descending and circular

chromatography?

20) Name the stationary phase in paper chromatography.

21) What is TLC?

22) Name a few compounds used as stationary phase in TLC.

23) What do you understand by the term visualising or spraying agent?

Give a few examples.

24) What is the visualising agent for amino acids? Discuss the chemistry

involved.

25) What is column chromatography?

26) What is Rf?

Experiment

Aim – To separate the given mixture of amino acids by ascending paper

chromatography and calculate the Rf of each component.

Learning objectives

A students learns

1) The meaning and importance of chromatography as a method of separation.

2) Terms – stationary and moving phase,, visualising and spraying agent, Retention

factor (Rf)

3) And understands the concept of distribution and partition.

4) How to separate a mixture of organic compounds.

5) Understands the chemistry of reaction of an amino acid with

ninhydrin( used as the visualizing agent)

Requirements

Apparatus Chemicals

Chromatography jar fitted with a cork

and a hook

Whatman filter paper strip( )

Capillary tube

Sprayer

Solvent- n- Butanol: acetic acid:water

(4:1:5)

Given solution of amino acids*

Spraying or visualizing agent: 1%

Ninhydrin solution

REACTION OF NINHYDRIN WITH AMINO ACIDS----IS TO BE GIVEN

HERE

Note

*A mixture of any of following: serine & isoleucine, glycine & aspartic acid,

alanine & leucine etc. can be used for best separation of the components of

mixture as their Rf values are different in the given solvent system.

2) The number of components in a given mixture of amino acids can also be

identified

a) by circular or descending paper chromatography.

b) By TLC

2) Identity or non identity of two samples can also be established by

By spotting the samples on the same chromatogram and compairing their Rf

value

Aim –To indentify the number of carbohydrates present in the given mixture

Requirements

Apparatus Chemicals

Same as in the above experiment Solvent – ( Butanol: Acetone: Water;

4:5:1) or (Methyl Ethyl Ketone: Acetic

Acid: Water; 3:1:1) or ( Butanol:

Acetic Acid: Water; 4:1:5)

Solution of carbohydrates**

Spraying or visualizing agent:

(1)Aniline Hydrogen

Phthalate (Spray & heat

for 4-5 minutes at 100

degree centigrade)

(2)Anisidine Phthalate

**: Any one of the following combination may be given for chromatography:

(Glucose and Fructose), (Glucose and Sucrose), (Fructose and Sucrose)