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 Lassaignes test.

Methods used for detection of extra elements present in the organic compounds Various methods used for detection of extra elements in the organic compounds are: 1. Lassaignes test for N, S and halogens 2. Beilstein test for halogens

Theory Lassaignes 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.
Heat Organic compound containing C, N, S, X + Na (X = Cl, Br, I)

NaCN+Na2S+NaX

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 Lassaignes 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 Lassaignes extract.

Test for nitrogen Nitrogen 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 Lassaignes 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.
Na4[Fe(CN)6] + Na2SO4 6NaCN + FeSO4 air, FeSO4 Fe2(SO4)3 3Na4 [Fe(CN)6] + 2Fe2(SO4)3 Fe4[Fe(CN)6]3 + 6Na2SO4 Ferriferrocyanide Prussian blue

Test for sulphur The sodium sulphide formed due to the presence of sulphur in the organic compound can be tested by any of the following tests: (a) The Lassaignes 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 Lassaignes 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 Lassaignes extract and addition of FeCl 3 solution (Leibigs test), a blood red coloration is obtained. N + S + C + Na NaSCN [Fe (SCN)3] + 3NaCl Ferric 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 halogens Silver nitrate test The Lassaignes 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 Lassaignes extract is acidified with dil. HNO 3 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.) 3NaSCN + FeCl3

 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.
Boil

NaCN + HNO3 Na2S + 2HNO3

Boil

NaNO3 + HCN 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 bromine Positive 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 Lassaignes extract is oxidized to t he 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 Apparatus Ignition tubes, a pair of tongs, china dish, wire gauze, funnel, test tubes, etc. Chemicals Organic 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 Lassaignes extract 1. 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 Lassaignes 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 Nitrogen 1. To approximately 1mL of Lassaignes 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 Lassaignes 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 Lassaignes 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 together Leibig 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 Lassaignes 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 Lassaignes extract, NaCN and Na2S are formed (instead of NaSCN) which can be tested as above. Test for halogens (a) Silver nitrate test with Lassaignes extract: If Nitrogen and/or Sulphur are absent: 1. Take approximately 1mL of Lassaignes 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 Lassaignes 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 Lassaignes 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 Lassaignes extract. (b) Layer test (for bromine and iodine): 1. Acidify approximately 1mL of Lassaignes 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. Note 1. 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: (2) Safety glasses must be worn. (3) Sodium must be kept away from water. (4) Organic compound must be added enough to react with whole of sodium in the fusion/ignition tube. (5) 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).

(6) Heating must be done over a small flame initially so that the organic compound does not get volatilized. (7) Distilled water must be used for preparation of Lassaignes extract. (8) A wire gauze should be used while immersing red hot ignition tube in distilled water contained in a china dish for protection against mishap. (9) A freshly prepared solution of sodium nitroprusside and ferrous sulphate should be used. (10) 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 Lassaignes extract and how is it prepared? 4. Why is it essential to prepare Lassaignes extract for the detection of extra elements in the organic compound? 5. Why is Lassaignes extract alkaline? 6. Give the chemistry of Lassaignes 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 Lassaignes extract gives negative test for nitrogen: Hydroxyl amine, hydrazine, ammonium chloride and ammonium sulphate. 11.Discuss the chemistry of Lassaignes test for sulphur. 12.Can sulphuric acid be used instead of acetic acid for the acidification of Lassaignes extract for lead acetate test for sulphur? 13.Why is it necessary to acidify the Lassaignes 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 Lassaignes extract for silver nitrate test for halogens? 19.Discuss the chemistry of the Layers 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 observation Lassaignes Test for Nitrogen, Sulphur and Halogens: S. No. 1. Experiment Test for nitrogen A few crystals of ferrous sulphate or 1mL of freshly prepared ferrous sulphate solution was added to 1mL of Lassaignes extract in a test tube and the mixture was boiled gently. Dilute sulphuric acid was added to the precipitate. Observation A green precipitate was formed. Inference

A Prussian blue or a deep green coloration observed.

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

OR Dilute sulphuric acid was added to the precipitate.

No blue coloration Nirogen absent or ppt. is obtained

2.

Test for sulphur Nitroprusside 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 Lassaignes extract in a test tube. OR 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 Lassaignes extract in a test tube. Lead acetate test: Acidified 1mL of Lassaignes extract with acetic acid in a test tube and then added 1mL of lead acetate solution.

Intense violet colouration observed.

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

No violet colour observed.

Sulphur absent.

A black precipitate was formed.

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

OR 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 Lassaignes extract in a test tube.

No black precipitate formed.

3.

Test for nitrogen and sulphur if present together: Leibig test: Acidified 1mL of Lassaignes A blood red extract with dilute HCl in a test colouration was tube and then added about 1mL of observed. ferric chloride solution.

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

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

No blood red coloration observed.

Both Nitogen and sulphur absent.

4.

Bright green flame Halogen(Cl, Br was observed. or I) present

AgNO3 test: Acidified the 1mL of Lassaignes 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 OR b) the pale yellow precipitate obtained which is partially soluble in ammonia solution

a) Chlorine is present.

b) Bromine is present.

Layer test: To 1mL of Lassaignes solution added 1 mL of concentrated 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.

OR c) deep yellow precipitate obtained which is insoluble in ammonia a) A colorless layer was observed. b) Yellowishorange color observed in the organic layer c) Violet colour observed in the organic layer

c) Iodine is present.

a) Chlorine may be present or halogens are absent. b) Bromine is present.

c) Iodine is present.

Beilstein test for halogens Introduction This 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 Lassaignes 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. Theory This 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

Procedure 1. 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 crystallization

The 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: Apparatus Test tubes, boiling tubes, funnel, filtering tube, filter button, Buchner funnel, Buchner flask, tongs, glass rod, etc. Chemicals Given 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

Precautions 1. 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.

Result Total 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 1C, 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 Kjeldahls 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 1C, 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 Kjeldahls flask for the determination of the melting point: Cleaning and drying of Kjeldahl flask 1. 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 liquids Generally three liquid compounds (i) concentrated sulphuric acid (ii) medicinal paraffin (iii) silicone oil (marketed as MS550 fluid) are be used as bath liquids in Kjeldahls 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 oil Silicone 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 noncorrosive liquid which can be safely heated upto 300 0C without decomposition or ignition.

Checking the calibration of the thermometer A 360C thermometer is normally used for determination of the melting point. The least count generally is 1C. The thermometer is calibrated by the manufacturer. For marking the thermometer, one of the procedure adopted is- The thermometer is kept in ice (for 0C mark) and in boiling water (for 100C mark) and then marked for rest of the temperatures up to 100C by dividing the length (between 0C and 100C mark) equally into 100. Similarly the thermometer is marked for temperatures from 100-360C. 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.

3.

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

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.

Requirements Apparatus Kjeldahls 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 Kjeldahls 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 Kjeldahls 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 Kjeldahls 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 1C, 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 properly 7. 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 Kjeldahls 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 1-2C 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 Kjeldahls 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 Kjeldahls 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 Kjeldahls 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 thermometer 9. 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 Kjeldahls 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 Kjeldahls 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 knowweather 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 Kjeldahls method (Siwoloboffs method) Theory Distillation method A pure liquid (which distills without decomposition) will have a sharp boilingpoint, 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, Kjeldahls 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 assembly Quick-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 Kjeldahls 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: Apparatus Chemicals Round bottom flask, still head, Given 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 150 0C, 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 SHOWN Notes:

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. Kjeldahls method (Siwoloboffs method) Requirements: Apparatus Kjeldahls flask

or

Chemicals beaker, Given sample,

concentrated

ignition tubes, a thin capillary tube, sulphuric acid. sand bath, clamp and clamp stand, thermometer fitted in the cork, dropper, rubber band, and Bunsen burner.

Procedure: 1. In Siwoloboffs 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 Kjeldahls 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 Kjeldahls 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 1-2C 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. 4.

The heating is not uniform. 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 Kjeldahls 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. : Siwoloboffs 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 Kjeldahls 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 Kjeldahls flask while determining the b. pt.? 10.Describe Siwoloboffs method of boiling point determination. 11.Describe distillation method of boiling point determination. 12.What are the advantages of distillation method over Kjeldahls 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.

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.

Chromatography

Adsorption (Solid stationary phase)

Partition (liquid stationary phase)

Liquid mobile phase

Gaseous mobile phase

Liquid mobile phase

Gaseous mobile phase

Thus depending on the nature of stationary phase and mobile phase, four types of combinations are possible i. ii. iii. iv. Liquid-solid Gas-solid Liquid-liquid 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 Covalent compounds of different chemical nature Volatile compounds or gaseous compounds Ions or ionic compounds Paper, TLC, column, ion exchange etc. Ionic compounds from covalent compounds Ion exchange Gas chromatography Adsorption Partition chromatography

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 ? chromatography

) remains similar to the paper

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) flask. 13) Transfer the liquid into the distillation flask and remove the solvent by Collect the fraction containing the compound in a separate conical

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) 13) What is column chromatography? 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 Chromatography jar fitted with a cork and a hook Whatman filter paper strip( ) Capillary tube Sprayer

Chemicals 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 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

Same as in the above experiment

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 crystallization The 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: Apparatus Test tubes, boiling tubes, funnel, filtering tube, filter button, Buchner funnel, Buchner flask, tongs, glass rod, etc. Chemicals Given 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

Precautions 8. 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.

Result Total 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 1C, 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.

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.

Chromatography

Adsorption (Solid stationary phase)

Partition (liquid stationary phase)

Liquid mobile phase

Gaseous mobile phase

Liquid mobile phase

Gaseous mobile phase

Thus depending on the nature of stationary phase and mobile phase, four types of combinations are possible v. vi. vii. viii. Liquid-solid Gas-solid Liquid-liquid 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 Covalent compounds of different chemical nature Volatile compounds or gaseous compounds Ions or ionic compounds Paper, TLC, column, ion exchange etc. Ionic compounds from covalent compounds Ion exchange Gas chromatography Adsorption Partition chromatography

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 ? chromatography

) remains similar to the paper

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) 12) Take a strip ( figure ?) of Whatman no 1 filter paper 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) 16) Cover the jar and allow the set up to stand undisturbed. 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) 17) 18) Coloured rings are observed if the components are coloured. Spray with locating agent, if no spots are visible 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) 16) Take a glass column with a nozzle at one end 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) 19) Clamp the column vertically on a stand. 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) flask. 27) Transfer the liquid into the distillation flask and remove the solvent by Collect the fraction containing the compound in a separate conical

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) 16) 17) Why it was called chromatography? How is the method used for separation of colourless compounds? 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) 21) 22) 23) Name the stationary phase in paper chromatography. What is TLC? Name a few compounds used as stationary phase in TLC. 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) 26) What is column chromatography? 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 Chromatography jar fitted with a cork and a hook Whatman filter paper strip( ) Capillary tube Sprayer

Chemicals 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 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

Same as in the above experiment

**: Any one of the following combination may be given for chromatography: (Glucose and Fructose), (Glucose and Sucrose), (Fructose and Sucrose)