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3 rd Year Organic Lab Manual


Page No.


1. Isolation of caffeine from tea


2. Isolation of euginol from clove


3. Isolation of lactose from milk powder


4. Isolation of lycopene from tomato


5. Isolation of chlorophyl from green leaves


6. Isolation of D-limonene from orange peel


7. Isolation of curcumin from turmeric



1. -napthaldehyde


2. Hippuricacid


3. Methyl orange


4. Thiamine hydrochloride catalysed synthesis of benzoin


5. Hydantoin synthesis



Experiment 1

Extraction of Caffeine from Tea

Purpose is to learn some of the basic techniques of organic chemistry: extraction, filtration, evaporation of a solvent and drying methods-in the context of working with a chemical known to all, caffeine.

Pure caffeine is a white, tasteless substance that makes up as much as 5% of the weight of tea leaves. By structure, caffeine is closely related to the purine bases, guanine and adenine, found in deoxyribonucleic acids (DNA).

guanine and adenine, found in deoxyribonucleic acids (DNA). A number of plants contain caffeine and its

A number of plants contain caffeine and its use as a stimulant predates written history. The origins of tea and coffee are lost in legend. In addition to being in tea leaves and coffee beans, caffeine is a natural constituent of kola nuts and cocoa beans. Cola soft drinks contain 14-25 mg of caffeine per 100 mL (3.6 oz), and a sweet chocolate bar weighing 20 g (0.7 oz) contains about 15 mg of caffeine. "Stay awake" preparations such as No Doz have caffeine as a main active ingredient. The caffeine content of tea leaves depends on the variety and where they were grown; most tea has 3-5% by weight. Coffee beans contain only about 2% caffeine by weight, yet a cup of coffee has about 3.5 times as much caffeine as does a cup of tea. How can this be? Coffee is usually boiled in its brewing or else ground extremely fine:

tea leaves are simply steeped in hot water for a few minutes Furthermore more ground coffee than tea is used to brew one cup of beverage. A cup of tea contains about 25 mg of caffeine. The biological action of caffeine includes cardiac and respiratory stimulation, and


it has a diuretic effect as well. Tea also contains a trace of the alkaloid theophylline, which is similar in structure to caffeine; it stimulates muscle action and relaxes the coronary artery. Theophylline also has veterinary applications as a diuretic and a cardiac stimulant.

Obtaining pure caffeine from tea requires a method for separating caffeine from the other substances found in tea leaves. Cellulose, the primary leaf component, poses no problem, because it is virtually insoluble in water. However, a large class of weakly acidic molecules called tannins also dissolve in the hot water used to dissolve the caffeine from tea leaves. Tannins are colored compounds having molecular weights between 500 and 3000 and phenolic groups that make them acidic. If calcium carbonate, a base, is added to tea water, calcium salts of these acids form in the tea solution. The caffeine can then be separated from the alkaline tea solution by a process of extraction using dichloromethane, an organic solvent in which caffeine readily dissolves. The calcium salts of the tannins remain dissolved in the aqueous solution. Flavinoid pigments and chlorophylls also contribute to the color of a tea solution. Although chlorophylls have some solubility in dichloromethane, the other pigments do not. Thus, the dichloromethane extraction of a basic tea solution removes nearly pure caffeine, which has a slight green color from the chlorophyll impurity.

After the extraction procedure, the organic solution of dichloromethane and caffeine is dried with an anhydrous inorganic salt. Crude caffeine is recovered as a solid residue by evaporation of the dichloromethane The solubility of caffeine in water at 20°C is 2.2 g per 100 mL, so there is no problem in keeping it in water solution while you filter off the spent tea leaves and calcium salts. Caffeine is far more soluble in dichloromethane: 10.2 g per 100 mL at 20% So this extraction takes advantage of distribution coefficient (k) of 4.6. To Conserve dichloromethane and time, we will settle for two 15 mL extractions of the aqueous tea solution. This method does not extract all the caffeine but yields more than enough for the purification step. The 10 g of tea that you boil with water should contain at least 300 mg of caffeine. You will be able to recover 10-30% of this amount.


A comment about filtering the boiled tea solution should be made before you begin. If it is filtered when it is too hot, messy bubbling occurs in the filtrate and some solution may be lost. Yet if it is filtered when it is too cool, the gelatinous material that separates on cooling will clog the pores of the filter paper. Fast, non retentive filter papers such as Schleicher and Schuell (S&S) No. 410 and Whatman No. 54 work well.

Place approximately 10 g of tea leaves in a 400-mL weighed (tared) beaker; record the mass of the tea leaves. If you use teabags, four bags should contain about 10 g of tea; remove the tea leaves from the bags and place the tea in the beaker. Add 4.8 g of. Calcium carbonate and pour 100 mL of water over the tea. Boil the mixture gently on a hot plate for 15 min, stirring every minute or two with a stirring rod.

Let the tea mixture cool to about 55 o C, then filter it, using vacuum filtration through S&S No. 410 or Whatman Filter paper. Pour the tea mixture in the Buchner funnel in two portions. If the filter paper clogs while the first portion is filtering, replace it with a fresh piece before filtering the remainder of the tea mixture. Cool the filtered solution to 15-20°C by adding a few ice chips. Set up a 125-mL separatory funnel and pour the cooled tea solution into the separatory funnel (be sure the stopcock is closed). Add 15 mL of dichloromethane to the funnel. Stopper the separatory funnel, hold the stopper firmly in place with your index finger, and invert the funnel. Open the stopcock to vent the vapors. Rotate the inverted funnel for 2-3 min, so that the two layers swirl together many times, opening the stopcock frequently to vent the funnel. Allow the layers to separate and then drain the dichloromethane layer into a 50mL Erlenmeyer flask. If a small emulsion layer is present at the interface between the organic and aqueous phases, add it to the Erlenmeyer flask. Cork the Erlenmeyer flask to prevent evaporation of the dichloromethane. Add 15 mL of fresh dichloromethane to the separatory funnel (still containing the tea solution) and repeat the extraction process. Again, allow the layers to separate and drain the dichloromethane layer, including any emulsion layer, into the Erlenmeyer flask containing the dichloromethane solution from the first extraction. Pour the tea solution out of the top of the separatory funnel into a beaker












dichloromethane solutions into the funnel; add about 20mL of water. Stopper the funnel, invert and rock it gently to mix the two layers. Some emulsion layer may be present at this point. If only a thin layer of emulsion exists at the interface between the aqueous phase and the dichloromethane solution, push a small piece of glass wool to the bottom of the dichloromethane layer with a large stirring rod. The glass wool will break the membranes of the emulsion. Drain the lower dichloromethane layer slowly into a clean, dry 50-mL Erlenmeyer flask. Add anhydrous magnesium sulfate to the dichloromethane solution. Cork the flask and allow the mixture to stand for at least 10 min, swirling the flask occasionally. Weigh (tare) a dry 50-mL Erlenmeyer flask on a balance that measures to 0.001 g. Place a fluted filter paper in a dry conical funnel and filter the drying agent from the dichloromethane solution collecting the filtrate in a tared 50 mL Erlenmeyer flask. Rinse the magnesium sulfate remaining in the flask with approximately 2 mL of dichloromethane and also pour this rinse through the funnel. Add a boiling stick or boiling chip to the flask containing the dichloromethane solution so that it boils without bumping Evaporate the dichloromethane on a steam bath or water bath heated on a hot plate in a hood. Alternatively, the dichloromethane may be removed by evaporation, using a stream of nitrogen, in a hood, or with a rotary evaporator. Continue the evaporation until a dry greenish residue of crude caffeine forms on the bottom of the flask. Weigh the flask and determine the mass of crude caffeine. Calculate the percent recovery. Cork the flask and store it in your laboratory drawer for purification a nd analysis.


Experiment 2 Steam Distillation: Isolation of eugenol from cloves

Purpose :

To perform a steam distillation using a microscale distillation apparatus and isolate a natural product i. e, eugenol from cloves.


O HO Eugenol

The boiling point of eugenol, an oil found in cloves, is 248 °C, but it can be isolated at a lower temperature by performing a co-distillation with water, this process is also know as a steam distillation. Eugenol is a phenyl propene, an allyl chain substituted guaiacol. It is a member of phenyl propanoids, a class of chemical compounds.

Since eugenol is not soluble in water, the concentration of the eugenol in the vapor over the boiling eugenolwater suspension does not depend on concentration of the eugenol. The relative amounts of eugenol and water in the vapor simply depend on the vapor pressures of the pure materials. The vapor pressure of water at 100 °C is 760 torr, and the vapor pressure of eugenol at 100 °C is approximately 4 torr; therefore, the vapour is roughly 0.5 % eugenol. (Note, the suspension boils when it’s vapor pressure is equal to the external pressure. Since both the eugenol and the water are contributing to the vapour pressure of the suspension, the suspension will boil before either pure substance would normally boil.) Since the distillate will contain both water and eugenol, the eugenol must be extracted from the water using an organic solvent. Once the eugenol is extracted into


an organic solvent, the organic layer is separated from the aqueous layer and dried. The eugenol is finally isolated by evaporation of the organic solvent.


1. Take about 50 g of clove in 500 ml of round bottom flask along with water that is

attached with distillation apparatus.

2. Distillation is to be performed on a heating mantel until 100 ml of condensate is


3. Extract the condensate into an organic layer (CCl 4 layer or DCM layer).

4. Add about 25 ml of 5% KOH to this organic layer and collect the aqueous layer.

5. Then acidify the aqueous layer with HCl and check it with litmus paper

6. Further, extract the overall solution with CCl 4 and add anhydrous sodium sulphate to

remove traces of water.

7. Evaporate the organic extract in the rotary evaporator to get the pure eugenol.

8. Finally, confirm the presence of phenolic OH group by the FeCl 3 test

Note: The initial addition of alkali should be sufficient such that eugenol present in the organic layer may totally get converted into its corresponding phenolate ion.


Experiment 3 Isolation of Lactose from Milk

Theoretical Background:

Milk is the most nutritionally complete food found in nature. All kinds of milk, human or animal, contain vitamins (principally thiamine VitB 1 , riboflavin VitB 2 , pantothenic acid VitB 5 , and vitamins A, B 12 , D), minerals (calcium,potassium, sodium, phosphorus, and trace metals), proteins(mostly casein), carbohydrates (principally lactose), and lipids (fats). Whole milk is an oil-in-water emulsion, containing its 3.9% fat dispersed as micronsized globules. The fat emulsion is stabilized by complex phospholipids and proteins that are adsorbed on the surfaces of the globules. Because the fat in milk is so finely dispersed, it is digested more easily than fat from any other source. The globules are lighter than water, thus coleasce on standing and eventually rise to the surface of the milk as cream. Vitamins A and D are fat-soluble substances and are thus concentrated in the cream. The fats in mil kare primarily triglycerides, which are esters of saturated and unsaturated carboxylic acids with glycerol, a tri-alcohol. There are three kinds of globular proteins in milk; caseins, lactalbumins, and lactoglobulins. Casein is a phosphoprotein, meaning that phosphate groups are attached to some of its amino acid side-chains. These are attached mainly to the hydroxyl groups of the serine and threonine moieties forming a type of alkyl phosphate. Casein exists in milk as the calcium salt, calcium caseinate. The salt has a complex structure which forms soluble micelles. Calcium caseinate has its isoelectric (neutrality) point at pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6. The pH of milk is about 6.6; thus, casein has a negative charge at this pH and is solubilized as a salt. If acid is added to milk, the negative charges on the outer surface of the micelles are neutralized and the neutral protein precipitates.

to milk, the negative charges on the outer surface of the micelles are neutralized and the


Albumins are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat. The second most abundant protein types in milk are the lactalbumins. Once the caseins have been removed, and the solution has been made acidic, the lactalbumins can be isolated by heating the mixture to precipitate them. A third type of protein in milk is the lactoglobulins. These are present in smaller amounts than the albumins and generally denature and precipitate under the same conditions as the albumins. The lactoglobulins carry the immunological properties of milk. They protect the young mammal until its own immune system has developed. When the fats and proteins have been removed from milk, the carbohydrates remain, as they are soluble in an aqueous solution. The main carbohydrate in milk is Lactose. Lactose, also called Milk Sugar, is not found in plants, and is one of the less sweet sugars. Lactose, being only about 1/6 as sweet as Sucrose, is the reason why milk has a rather bland taste. This sugar is only formed in the mammary glands of lactating mammals and is the only carbohydrate that mammals can synthesize.


Acetic Acid, CH 3 COOH Ethyl ether, C 4 H 10 O Ethanol, C 2 H 5 OH Milk


Isolation of Lactose from Milk Weigh out 10g of non-fat powdered milk in a weighing boat. Dissolve the milk in 100mL of warm water in a 250mL beaker. Heat the milk solution to 40 o C. Add 6mL of 10% acetic acid to precipitate out the casein, and stir the mixture slowly and briefly. Avoid adding excess of acetic acid. Work the casein into a mass and remove it with a stirring rod or spoon. Immediately add 2.5g of powdered calcium carbonate. Stir the mixture thoroughly. Heat the mixture almost to boiling for about 10 minutes with constant


stirring. This process will precipitate the remaining proteins. Filter the hot mixture and collect the filtrate in a 250 mL beaker. Concentrate the filterate to 10mL by evaporating through boiling, and then add 50 mL of 95% ethanol. Heat the solution up to 70 o C to get homogenius mixture (ethanol b.p. 78 o C). Filter the warm ethanol solution and collect the filtrate in a 125mL conical flask. Label and stopper the flask and place it in your lab drawer until the next lab period. Filter off the crystals of lactose. Allow them to dry for one hour. Weigh the crystals. Compare the percentage of recovery of lactose with the percentage of carbohydrate as reported on the nutritional label of the powdered milk used.


Experiment 4 Isolation of Lycopene From Tomato Paste


Lycopene, the red pigment of the tomato, is a C40-carotenoid made up of eight Isoprene units; making it a tetraterpene.

made up of eight Isoprene units; making it a tetraterpene. β -Carotene, the yellow pigment of

β-Carotene, the yellow pigment of the carrot is an isomer of Lycopene in which the double bonds at C1-C2 and C'1-C'2 are replaced by bonds extending from C1 to C6 and from C'1 to C'6 to form rings, and is also a constituent of the tomato.

to form rings, and is also a constituent of the tomato. Each of these compounds is

Each of these compounds is classified as a Carotenoid.


Carotenoids are organic pigments that are naturally occurring in the chloroplasts and chromoplasts of plants and some other photosynthetic organisms like algae, some types of fungus and some bacteria. There are over 600 known carotenoids; they are split into


two classes, xanthophylls (which contain oxygen) and carotenes (which are purely hydrocarbons, and contain no oxygen). Carotenoids in general absorb blue light. They serve two key roles in plants and algae: they absorb light energy for use in photosynthesis, and they protect chlorophyll from photodamage.

Isolation through Column Chromatography:

We will isolate Lycopene from tomato paste, which as noted above, contains high levels of these pigments, using Column Chromatography. Like other forms of chromatography, Column Chromatography is based on a two phase system where the stationary phase is a column of adsorbant and the mobile phase is a liquid eluent. The theory of column chromatography is analogous to that of thin-layer chromatography. The most common adsorbents, silica gel and alumina, are the same ones used in TLC. The sample is applied to the top of the column. The eluent, instead of rising by capillary action up a thin layer, flows down through the column filled with the adsorbent. Just as in TLC, there is an equilibrium established between the solute adsorbed on the silica gel or alumina and the eluting solvent flowing down through the column. Under some conditions, the solute may be partitioning between an adsorbed solvent and the elution solvent, the partition coefficient, just as in the extraction process, determines the efficiency of separation chromatography. The partition coefficient is determined by the solubility of the solute in the two phases. In general, the amount of alumina or silica gel used should weigh at least 30 times as much as the sample, and the column, when packed, should have a height at least 10 times the diameter. The density of silica gel is 0.4 g/mL and the density of alumina is 0.9 g/mL, so the optimal size for any column can be calculated. Uniform packing of the chromatography column is critical to the success of this technique. The sample is adsorbed onto a small quantity of adsorbent as a pure liquid or, if it is a solid, as a very concentrated solution in the solvent that will dissolve it best, regardless of polarity. As elution takes place, this narrow band of sample will separate into several bands corresponding to the number of components in the mixture and their relative polarities and molecular weights. It is essential that the components move


through the column as a narrow horizontal band in order to come off the column in the least volume of solvent and not overlap with other components of the mixtures. Therefore, the column should be vertical, and the packing should be perfectly uniform, without voids caused by air bubbles. The preferred method for packing silica gel and alumina columns is the slurry method, whereby a slurry of the adsorbent and the first eluting solvent is made and poured into the column. When nothing is known about the mixture being separated, the column is prepared in ligroin or hexanes, the least polar of the eluting solvents. We will extract crude Lycopene from tomato paste and apply the extract to our column. Tomato paste, as a source for our Lycopene, has the advantage that its Lycopene concentration is significantly higher, gram for gram, than that of a ripe tomato. The main drawback of using tomato paste is that trans-Lycopene may isomerize during the cooking process during which the paste is produced. We will then pack a column for purifying the Lycopene. We will use neutral Alumina (Grade II-III), a fairly active adsorbant, to separate the Lycopene from other tomato paste pigments. Note:

Lycopene, with its 13 double bonds, is attracted to alumina more strongly than are β- carotene and related carotenes, which have 11 to 12 double bonds. Therefore, the yellow carotene band will move down the column faster than the orange-red lycopene band. Yellow xanthophyll pigments will trail behind the lycopene band because they contain polar hydroxyl groups that are strongly attracted to Alumina.

will trail behind the lycopene band because they contain polar hydroxyl groups that are strongly attracted



Take 10 gm of tomato paste in 25 ml of ethanol (95 %) and 50 ml of DCM in a 250 ml of round bottom flask. Reflux the resultant solution for 10 min with constant stirring. Filter the solution and discard the insoluble part. Wash the filtrate with saturated NaCl solution (100 ml) using separating funnel. Transfer the organic part in a round bottom flask before drying over anhydrous sodium sulphate. Remove the organic solvent using rotary

evaporator to obtain solid lycopene in crude form. The solid contains mixture of - carotene, lycopene and small portions of xanthophylls. Separate the lycopene using column chromatography with hexane containing 2% EtOAc as eluting solvent. The - carotene comes first with eluent followed by lycopene. Collect the eluent containing lycpoene and evaporate the solvent. Note down the yield of resultant solid and purity.


Experiment 5 Isolation of Chlorophyll from Spinach/Green Leaves


Chlorophylls are the green pigments that act as the principal photoreceptor molecules of plants. They are capable of absorbing certain wavelengths of visible light and then convert them into chemical energy. Two different forms of chlorophyll can be found in plants and they are chlorophyll A and B. In this experiment, we are trying to isolate the chlorophylls from the spinach leaves by means of TLC. The leaves of plants contain a number of colored pigments generally falling into two categories, chlorophylls and carotenoids. The green chlorophylls a and b, which are highly conjugated compounds capture the (non green) light energy used in photosynthesis.

Spinach leaves contain chlorophyll a, chlorophyll b and -carotene as major pigments as well as smaller amounts of other pigments such as xanthophylls. The xanthophylls, which are oxidized versions of carotenes, and pheophytins, which look like chlorophyll except that the magnesium ion is replaced by two hydrogen atoms.

look like chlorophyll except that the magnesium ion is replaced by two hydrogen atoms. Chlorophyll A

Chlorophyll A


look like chlorophyll except that the magnesium ion is replaced by two hydrogen atoms. Chlorophyll A
look like chlorophyll except that the magnesium ion is replaced by two hydrogen atoms. Chlorophyll A

Chlorophyll B

Experimental Procedure:

Part I: Extraction

Clean some spinach leaves with water and dry it with a paper towel. Cut 2g of spinach leaves into small pieces with scissors and put into the mortar. Add 3 mL of acetone into a mortar and grind the leaves with the pestle. Filter the solution through a funnel using cotton at narrow end in to test tube (A) and squeeze the cotton to get more extract.

Pour 3 mL of acetone into the mortar and grind the residue with the pestle again. Pour the liquid part into the above test tube (A) through the funnel. Then pour 3 mL of acetone and 5 mL of hexane into the mortar and grind the residue again and then filter the extract into the another test tube (B). The residue left over should be very pale green.

Add 3 mL of brine into the test tube (B). Put on the cap tightly to test tube B and shake the test tube vigorously. Open the screw cap of the test tube occasionally to release the gas pressure while shaking. Then set the tube back on the test-tube rack and allow the mixture to separate. The top layer should be a dark green and the bottom layer should be a clear light green.

green and the bottom layer should be a clear light green. If emulsion is hard to
green and the bottom layer should be a clear light green. If emulsion is hard to

If emulsion is hard to separate, add some brine to facilitate the separation. By using a dropper, separate and collect the organic layer to a clean screw-cap test tube (C).

Add enough anhydrous sodium sulphate to the solution in test tube (C) that contains the chlorophyll for drying the solution. Then filter the solution with a filter paper into a 25-mL conical flask.

the chlorophyll for drying the solution. Then filter the solution with a filter paper into a


Column Chromatography:

Use a mixture of acetone and hexane in the volume ratio of 2:8 as an elutant and do the column chromatography by packing the column with silica gel. Collect both the pigments separately and evaporate using rotary evaporator. Perform TLC to check the purity of the pigments.


Since the experiment employs a highly non-polar solvent (acetone-hexane mixture), the pigments that are least polar (carotenes) will be best dissolved in the non-polar solvent and will thus have the largest R f value. Accordingly, the first fraction will be containing carotenes. Collect this fraction until no more carotene is seen (verify by TLC). The second and third fractions will be Chlorophyll A and B respectively which should be collected separately. The order of the fractions depends on the polarity of the molecules, the less polar molecules elute first and the most polar one elutes last.


Experiment 6 Isolation of Limonene from Orange Peel


To extract limonene, a terpene, from orange peel via steam distillation.


Limonene (1-methyl-4-prop-1-en-2-yl-cyclohexene) is an unsaturated hydrocarbon, belongs to the class of terpenoids. It is a colorless oily liquid with the smell of oranges. Its molecular formula is C 10 H 16 and its boiling point is 176 °C. Limonene is a chiral molecule with two optical isomers (enantiomers). The major biological form d-limonene, (R)-enantiomer, is used in food manufacture and medicines. It is also used as a fragrance in cleaning products, a botanical insecticide, and also as potential biofuel due to its flammability. The (S)-enantiomer, l-limonene, is also used as a fragrance but has a piney, turpentine odour.

also used as a fragrance but has a piney, turpentine odour. Limonene Steam Distillation: The distillation


Steam Distillation:

The distillation of a homogeneous binary mixture produces a vapor to which each component in the mixture contributes a part. The distillation of a heterogeneous binary mixture, on the other hand, produces a vapor mixture that is only dependent upon the temperature. Thus, if a mixture is composed of water and a high-boiling, water- immiscible substance, the mixture will boil near but somewhat below 100 o C. Most of the distillate will be water, but a separable portion of it will be the immiscible component, usually in a high state of purity. The process just described, called steam d istillation, is an


excellent technique for the isolation of many water-insoluble compounds that are unstable at temperatures near their boiling points, or compounds difficult to isolate or purify in any other way. Steam distillations may be carried out in the following ways. The compound to be separated and water are simply placed in a conventional distillation apparatus and the distillation collected. This is called direct steam distillation. The distillate, which appears as a heterogeneous mixture, may be either two liquid layers or a solid and water. A separation is easily effected by way of the separatory funnel or by simple separation. In the present experiment we will isolate by way of direct steam distillation a sample of limonene from a few oranges. Limonene is an essential oil made up of two isoprene units, that belongs to a class of compounds, known as terpenes.

units, that belongs to a class of compounds, known as terpenes. Steam Distillation Set-up For Isolation

Steam Distillation Set-up For Isolation of Limonene



Cut orange peel into small pieces and place them in a 250 ml round bottom flask. Add around 100 ml of distilled water to it. Set the distillation set up and then heat the flask so that the distillation proceeds at a steady rate. Add 1 or 2 boiling chips to avoid violent boiling. Periodically add 10-20 ml of distilled water to the distillation flask to maintain a constant volume. Continue the distillation until about 30 ml of distillate has been collected. By this time, an oily layer may be seen on the surface of the collected distillate, which should be of about 1 ml. If no such layer is formed, it may be necessary to collect a larger volume of the distillate and concentrate it. Transfer the distillate to the separatory funnel and collect the organic layer (DCM layer) containing the limonene. Evaporate the residual DCM to isolate the oily mass, Limonene.


Experiment 7 Isolation of Curcumin from Turmeric


Turmeric is commonly known for its medicinal values in the Indian traditional system of medicine. Turmeric has been used as “ayurvedic medicine” in particular for its antiseptic, wound healing, and anti-inflammatory activities. Curcumin, dimethyoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC) are dietary photochemicals which are obtained from dried rhizomes of the turmeric plant (curcuma longa). Curcumin, DMC and BDMC are together known as curcuminoids which are main coloring substance in curcuma longa. The value of the turmeric products is based on their curcuminoid content. Quantitative estimation of curcuminoids can be carried out photometrically based on its absorbance at 420 nm .The principal coloring components of curcumin exhibit a keto- enol tautomerism and antioxidative properties.

Me O


O H O Curcumin

OM e











Curcumin is the principle component of turmeric, which is a member of the ginger family. Curcumin can exist in two tautomeric forms, keto and enol. The enol form is energetically more stable in solid phase and in solution.



Take 20 gm of ground turmeric, add 50 ml of DCM and reflux the solution for about 1 hour with continuous stirring. Filter the resultant solution using suction pump and concentrate the filtrate on a hot water bath. Triturate the reddish yellow oily residue with 20 ml of hexane and then collect the resulting solid by suction filtration. Dissolve the resultant crude precipitate in minimum amount of DCM and load it into a column, packed with silica gel. Run the column with 3% MeOH/DCM mixture to collect the curcumin.




Preparation Of β-Hydroxy naphthaldehyde

Aim: Preparation of β-Hydroxy naphthaldehyde from β-naphthol by Reimer-Tiemann Reaction.


Reimer-Tiemann Reaction is a chemical reaction used for ortho-formylation of phenols. The attacking moiety in this reaction is dichloro carbene (:CCl 2 ), an electrophilic reagent produced by the treatment of chloroform with bases. Dichloro carbene reacts at the ortho position of phenolate ion to give the dichloromethyl substituted phenol. After base hydrolysis, the desired product is formed. The reaction is highly exothermic in nature and the initiation point of the reaction is visualized through formation of a deep blue colour.

Reaction Mechanism:

Chloroform (1) is deprotonated by strong base (normally hydroxide) to form the chloroform carbanion (2) which will quickly alpha-eliminate to give dichlorocarbene (3); this is the principal reactive species. The hydroxide will also deprotonate the phenol (4) to give a negatively charged phenoxide (5). The negative charge is delocalised into the aromatic ring, making it far more nucleophilic and increases its ortho-selectivity. Nucelophilic attack of the dichlorocarbene from the ortho position gives an intermediate dichloromethyl substituted phenol (7). After basic hydrolysis, the desired product (9) is formed.


Reaction conditions: Hydroxides are not readily soluble in the chloroform, thus the reaction is generally

Reaction conditions:

Hydroxides are not readily soluble in the chloroform, thus the reaction is generally carried out in a biphasic solvent system. In the simplest sense this consists of an aqueous hydroxide solution and an organic phase containing the chloroform. The two reagents are therefore separated and must be brought together for the reaction to take place.


In a round-bottom flask fitted with a reflux condenser, dissolve β-naphthol (5 g) by shaking it with a solution of sodium hydroxide (10 g), dissolved in a mixture of alcohol (20 ml) and water (20 ml). Add Chloroform (3.5 ml) through condenser into it dropwise over a period of 15-20 min. Shake the contents and continue reflux for about 45-50 min. Distill off the alcohol and acidify the residue with conc. HCl. Extract the desired product with ether. Evaporate the ethereal solution to get the residue. Recrystallize the residue with ethanol. Report the yield and mp.


Synthesis of Hippuric Acid

Aim: To synthesize hippuric acid from glycine and benzoyl chloride.

Chemicals Required:

Benzoyl Chloride


Sodium Hydroxide Solution

Conc. HCl

Apparatus Required:

Conical Flask

Measuring Cylinder

Erlenmeyer Flask

Buchner Funnel



Synthesis of hippuric acid starts with glycine, an amino acid commonly found in proteins. The reaction of glycine with benzoyl chloride in the presence of an alkaline catalyst yields the amide, benzoylglycine (hippuric acid). When the amino acid glycine is in its zwitter ionic form (dipolar form), the nitrogen of glycine is no longer nucleophilic in nature. Treatment of zwitter ionic glycine with NaOH converts the amino acid to its respective nucleophilic form and the amino acid bearing the electron pair readily attacks benzoyl chloride to initiate amide formation. Loss of the elements of the HCl yields a carboxylate salt, which when acidified produces hippuric acid.


Reaction: O Cl O NH CH 2 COOH i) NaOH COOH ii) HCl N H
i) NaOH
ii) HCl
N H 2


Dissolve 2.1 g of glycine in 22 ml of H 2 O and 3 ml of 6N sodium hydroxide solution and mixed well. Add 3.1 ml of benzoyl chloride to it, further followed by addition of 3 ml of 6N NaOH. Requisite amount of NaOH is required to make the pH alkaline. Monitor the pH of the solution with a litmus paper. The final pH should be alkaline and colour of the solution yellow. Add conc. HCl to this solution to obtain a white precipitate. Filter the ppt. using a Buchner funnel and then keep it for drying.


Synthesis of Methyl Orange

In this experiment you will prepare methyl orange, an azo dye that forms beautiful orange crystals and is used as an acid-base indicator (Figure 1). The anion form is yellow and the acid form is red.

1). The anion form is yellow and the acid form is red. You will synthesize methyl

You will synthesize methyl orange from sulfanilic acid and N,N-dimethylaniline using a diazonium coupling reaction just like the one you saw in the previous experiment in the nitrous acid test for primary aromatic amines. The overall reaction is shown in Figure 2 and the mechanism is shown in figure 3.

is shown in Figure 2 and the mechanism is shown in figure 3. The first step

The first step is simply an acid base reaction. In order to dissolve the sulfanilic acid in the aqueous solution we add sodium carbonate. Then it form the diazonium salt. When we add the HCl, the nitroso ion is formed from sodium nitrite and this reacts with the amine to form a nitrosoammonium adduct that loses water under the acidic conditions after proton transfer. This gives the diazonium salt. Aromatic diazonium salts are stable at low


temperature. The terminal nitrogen of the diazonium salt is very electron defic ient. It can be attacked by good nucleophiles. We dissolve the dimethylaniline in acetic acid. This forms the dimethylaniline acetate salt which is soluble in water but not nucleophilic. Slow addition of sodium hydroxide neutralizes the protonated amine of the N,N- dimethylaniline acetate salt in situ and the dimethylaniline becomes a good nucleophile due to the activating effect of the dimethylamine substituent. Attack is in the para position due to hindrance at the ortho position by the bulky dimethylamine substituent.

the ortho position by the bulky dimethylamine substituent. Apparatus: i. Conical flask ii. Beaker iii. Measuring


i. Conical flask

ii. Beaker

iii. Measuring cylinder

iv. Vacuum filter

v. Round bottom flask

vi. Hot plate


Chemical required:

i. Sulphanilic acid

ii. Sodium carbonate (Na 2 CO 3 )

iii. Sodium nitrite (NaNO 2 )

iv. Conc. HCL

v. Dimethyl amine

vi. Glacial acetic acid


Diazotized Sulfanilic Acid

1. Add 4.8g of Sulphanilic acid in 50 ml of 2.5% Na 2 CO 3 in a coical flask and heat it

until it dissolves.

2. Cool the filtrate to 15 0 C, add 1.9 g of sodium nitrite, and stir until solution is complete.

3. Pour this mixture, while stirring, into a 100-mL beaker containing 25 g of crushed ice

to which 5 mL of concentrated hydrochloric acid have been added; add the HCl dropwise to maintain a temperature of 0-5 o C. The diazonium salt of sulfanilic acid should soon separate as a finely divided white precipitate. Keep this suspension cooled in an ice bath until it is to be used.

Methyl Orange

4. In another R.B flask mix together 3.2 mL of dimethylaniline and 2 mL of glacial acetic

acid to dissolve amine as its salt.

5. Add this solution dropwise to the cooled suspension of diazotized sulfanilic acid in the

100-mL beaker. Stir the mixture vigorously. In a few minutes, a red precipitate should form. Keep the mixture cooled in an ice bath for about 15 minutes to ensure completion of the coupling reaction.



Add 35 mL of a 10% aqueous sodium hydroxide (NaOH) solution. Do this slowly and

with stirring, as you continue to cool the beaker in an ice bath. Check with litmus or pH

paper to make sure the solution is basic. If it is not, add more base.

7. Heat the mixture to boiling with a Bunsen burner for 10 to 15 minutes to dissolve most

of the newly formed methyl orange. When all (or most of it) the dye is dissolved cool the mixture in an ice bath. The methyl orange should recrystallize. Filter using a Buchner

funnel. (If ppt did not appear add 2.5 g of sodium chloride, dissolve and cool again).

8. To purify the product, transfer the filter cake and paper to a large beaker containing

about 40 mL of boiling water. Maintain the solution at a gentle boil for a few minutes,

stirring it constantly. Note: Not all the dye will dissolve, but the salts with which it is contaminated will dissolve.

9. Remove the filter paper and allow the solution to cool to room temperature. Cool the

mixture in an ice bath, and when it is cold, collect the product by vacuum filtration, using a Buchner funnel. Allow the product to dry, weight it, and calculate the percentage yield.

Note: The product is a salt. Since salts do not generally have well-defined melting points, the melting-point determination should not be attempted.


Thiamine hydrochloride catalyzed synthesis of benzoin

Chemicals Required: Benzaldehyde 5 ml; Thiamine hydrochloride 0.88 g Sodium hydroxide 2.5 ml (2 M); Ethanol - 8 ml Introduction The reaction of two moles of benzaldehyde to form a new carbon-carbon bond is known

as the benzoin condensation. It is catalyzed by two rather different catalysts, cyanide ion and the vitamin thiamine, which on close examination are seen to function in exactly the same way.

examination are seen to function in exactly the same way. Non-green Component: Involves the use of

Non-green Component:

Involves the use of highly poisonous sodium cyanide Green way:

the use of highly poisonous sodium cyanide Green way: A number of biochemical reactions bear a

A number of biochemical reactions bear a close resemblance to the benzoin condensation

but are not, obviously, catalyzed by the highly toxic cyanide ion. Some 30 years ago Breslow proposed that vitamin B1, thiamine hydrochloride, in the form of the coenzyme thiamine pyrophosphate, can function in a manner completely analogous to cyanide ion in promoting reactions such as the benzoin condensation. In the scheme below, the resonance-stabilized conjugate base of the thiazolium ion, thiamine, and the resonancestabilized carbanion that it forms, are again the keys to the reaction. Like the cyanide ion, the thiazolium ion has just the right balance of nucleophilicity, ability to stabilize the intermediate anion, and good leaving group qualities. The importance of thiamine is evident in that it is a vitamin, an essential substance that must be provided in


the diet to prevent beriberi, a nervous system disease. In the reactions that follow, cyanide ion functions as a fast and efficient catalyst, although in large quantities it is highly toxic. The amount of potassium cyanide used in the present experiment (15 mg) is about eight times less than the average fatal dose, a difference that underlines the advantage of carrying out organic experiments on a microscale.

of carrying out organic experiments on a microscale. The thiamine hydrochloride (0.88 g) was dissolved in

The thiamine hydrochloride (0.88 g) was dissolved in water (about 2.5 ml) in a 50 ml round bottom flask. Ethanol (95%, 8 ml) was added and the solution was cooled by swirling the flask in an ice water bath. Meanwhile, sodium hydroxide solution (2.5 ml) was cooled in a small conical flask in an ice bath. Then over a period of about 10 min the sodium hydroxide solution was added drop wise to the thiamine solution. Fresh benzaldehyde (5 ml) was added to the reaction mixture. The mixture was heated gently on a water bath for about 90 min. Do NOT let the temperature go above 65°C at any time during the experiment as this causes undesirable side-reactions. The mixture was cooled to room temperature and then in ice bath to induce crystallization of the benzoin. If product is separated as oil, the mixture was reheated until it was once again homogeneous. Then it was allowed to cool more slowly than before. Scratching of the


flask with a glass rod may induce crystallization. Cap and set the vial in your locker until the next lab period to work-up the reaction. Caution:

Benzaldehyde used in the experiment should be free of benzoic acid

Thiamine hydrochloride should be kept in refrigerator when it is not in use. Green Context:

Hazardous and poisonous cyanide ion is replaced by thiamine hydrochloride.

Reaction is effected at a lower temperature.


Synthesis of 5, 5ʼ- Diphenylhydantoin

Apparatus Required:

Round Bottom Flask (250 ml) Reflux condenser Heatimg mantle Vacuum filtration apparatus Funnel Beaker

Chemicals Required:

Benzil Ethanol (95%) KOH Sulphuric acid (6N) Urea


The application of organic chemistry to the large scale commercial production of biologically active medicinal agents has in many cases resulted in the discovery of new and potentially useful reactions or reaction products. The synthesis of hydantoins, such as 5,5’-diphenylhydantoin (phenytoin), from their corresponding 1,2-diketones illustrates one such novel reactin that avoids the use of sodium cyanide characteristic of more classical methods of hydantoin synthesis.The mechanism closely resembles that of the benzyl-benzilic rearrangement, except that instead of using hydroxide or alkoxide anion as nucleophile, urea is used to generate ureide intermediate, which undergoes to give hydantoin.





1. 10.1g (0,05 mol) of benzil and 6.0 g(0.1 mol) of urea were dissolved in 100 ml of

ethanol in a 250 ml round bottom fkask.

2. To the above solution, a solution of 16.5g (0.3 mol) of potassium hydroxide in 20 ml

of water was added.

3. The mixture was refluxed for 2.5 hours.

4. Flask was cooled and the solids were filtered off.

5. Filtered liquid was cooled in an ice-water bath and slowly acidified with 6N sulfuric

acid, during which hydantoin was precipitated.

6. The product is filtered and dried to yield the respective phenytoin.