Lecture 21 Different methods of secondary metabolite, essential oil extraction and estimation Distillation Distillation, as the most important

process for obtaining essential oils, needs attention first. It may be defined as the "separation of the components of a mixture of two or more liquids by virtue of differences in their vapour pressure" (i) Water Distillation When this method is employed, the material to be distilled comes in direct contact with the boiling water. It may float on the water or be completely immersed, depending on its specific gravity. The water is boiled by application of heat by any of the usual methods, i.e., direct fire, steam jacket, closed steam coil, etc. Through this method, powdered materials like almond and others like rose petals and orange blossoms are distilled. Disadvantages of Water Distillation 1. If the temperature is high, there will be browning of the essential oil. 2. In this method, certain compounds like esters are partly hydrolyzed. 3. It requires a greater number of stills, more space and more fuel. 4. It demands considerable experience and familiarity to successfully use this method. (ii) Water and Steam Distillation In this case, the plant material is supported on a perforated grid or screen inserted at some distance above the bottom of the still, and the lower part of the still is filled with water to a level below the grid. The water is heated. The typical features of this method are: first, that the steam is always fully saturated, wet and never super-heated; second, that the plant material 'is in contact with the steam only and not with the boiling water. This method gives best results with materials like seeds and roots. Disadvantages of Water and Steam Distillation 1. To obtain the best results, it is necessary that the charge should be in homogenous individual pieces. 2. If the material is extremely wet, it results in lumping or agglomeration of the charge. 3. It takes a comparatively long time for distillation. (iii) Steam Distillation Distillation is the process by which essential oils are extracted from aromatic crops. This can be done in two ways: In this process, steam generated in a boiler is passed through steam pipes into distillation tank which is filled with harvested aromatic crop. Pressure, temperature and amount of the steam can be regulated as per requirement; thereby time of distillation can

also be varied. The steam converts the essential oil in the aromatic plants into oil vapour and both these pass through delivery pipe into condenser where due to the effect of circulating cold water, steam and oil vapour condense and the resulting mixture of water and essential oil is collected in the receiver of separator. Since the density of oil is relatively lesser than water, the oil floats on water and can easily be taken out from the separator through the outlet provided specially for taking out oil. This process is used in large scale production of essential oils from aromatic crops. The equipment (boiler, distillation tank, condenser and separator) needed for this process is expensive and needs technical personnel to run the boiler. This distillation unit consists of the following parts. a. Furnace and Chimney The furnace is constructed with bricks and mud and is used for heating the distillation tank kept over it. This can be fired using fire wood or exhausted plant material left after distillation or weeds. The chimney is made of mild steel and is useful for carrying the smoke coming out from furnace. b. Distillation Tank or Retort This is made of mild steel and is either cylindrical or rectangular in shape. The capacity of the tank is fixed based on the requirement. A special mesh fixed in it divides it into two parts and facilitates filling of water below and plant material above the mesh. A fixed number of G.I. pipes are welded at the bottom for increasing fuel efficiency. c. Condenser The outer shell and legs are made of mild steel and the tubes inside the shell are made of aluminium. This is connected to the distillation tank with the help of a delivery pipe. During distillation, cold water circulates in the condenser. The main purpose of the condenser is to condense the mixture of steam and oil vapour into water and essential oil. d. Receiver or Separator This is made of aluminium and is used to collect the mixture of water and oil coming out from the condenser. Separate outlets are provided for overflow of excess water and for taking out essential oil. e. Storage and packing of Essential oil Care is taken to ensure that the essential oil does not contain any water before storage. Amber colored bottles are convenient for small quantities. For large quantities. Steel or aluminium drums are widely used. The oils are left to stand for sometime so that water can settle down. If the oil is still turbid, a small amount of common salt is added and the oil filtered. The containers are filled up to the brim, tightly capped and stored in

a cool, dry, dark place. Exposure to air, light and water causes deterioration of the quality of essential oil. Qualities expected from steam distillation unit 1. Gives higher yield of Oil and more profit 2. Boiler consumes very less wood and requires minimum maintenance. 3. Gives Best Quality of oil which can be sold easily & at higher price. 4. Latest Technology and heavy duty material for higher safety 5. Can be operated with minimum labour force 6. Working in different parts of the Country. Aromatic oil distillation plant Capacity of 2 Distillation vessels Model a Commercial Model b Standard Model c Standard Model i.e with SS Vessels, top cover & vapour pipe 0.5 Ton Rs. 75,000/Rs.1.00,000/Rs.1,35,000/1.0 Ton 90,000/1,65,000/2,75,000/2.0 Ton ----------3,50,000/5,25,000/-

Scope of supply: Item nos. 1 to 6 listed below for both Commercial and Standard Model. 1) Distillation Vessels in MS 2 Nos. 2) Conical Top cover with Vapour pipe 1 No. 3) Water Boiler 1 No. 4) Stainless Steel tube Condenser 1 No. 5) Oil separator 1 No. 6) Pipe lines & Valves 1 Set. B) Auxillary items for the plant SNo. 1. 2. 3. 4. 5. Items Channel Base frame + Lifting Structure Trolley + Chain block MS Cages (4 Nos.) Cooling Tower (With Pump & Piping) 2,000 Its Water Circulation Tank 0.5 Ton 27,000 8,000 16,000 25,000 17,000 Rs.7,000 5 Acres 1.0 Ton 39,000 9,000 22,000 38,000 17,000 15,000 5-20 acres 2.0 Ton 40,000 10,000 30,000 45,000 17,000 8,000 20 Acres & more

C) Erection and commissioning: D) Plant recommended for crop area:

Advantages of standard model over standard model 1. Highest yield of Oil – Resulting in Additional Revenue, day after day. 2. Best Quality of Oil – Gets you very good price. 3. Low consumption of Fuel (about 250 Kg. to 300 Kg. dry Wood for 1.0 Ton Grass) 4. Safe & Trouble Free operations – suitable even for Remote Locations. 5. Robust Engineering with Heavy Metal thickness for Long Life. 6. Completely Integrated – Easy to Operate with 2 Labourers. 7. Based on Advanced Design & Fabricated as per GMP Standards. Solvent Extraction Process and other methods • Solvent Extraction process • Enfleurage or cold fat extraction • Maceration or hot fat extraction • Expression 1. Solvent Extraction process Although solvent extraction is a recent process in essential oil production, it had been as early as 1835, it was felt that volatile solvents could be used to extract the essences from flowers more conveniently than by maceration. This method once developed was quickly adopted for processing all types of perfumes from the flowers that do not continue to produce fragrance once they are picked. Principle The principle of extraction with volatile solvents is simple. Fresh flowers are charged into especially constructed extractors at room temperature and treated carefully with purified solvent usually petroleum ether/Hexane. The solvent penetrates the flowers and dissolves the natural flower perfume together with some waxes and other albuminous and colouring matters. This solution is subsequently pumped into an evaporator and concentrated at a low temperature. After the solvent is completely driven off in vacuum, the flower oil is obtained. Since no heat is applied in the method at any stage, the oil is saved from the harmful effects of higher temperatures and therefore more purely represent the natural perfume as originally present in the flowers. The flowers obtained from commercial flower crops like jasmines, rose and tuberose in addition to their conventional uses are now being used for the extraction of the floral concrete or aromatic oil which are needed by the high cost perfume industry. The essential oil present in these flower crops can not be separated by simple steam distillation. Hence the solvent extraction method is practiced in which the odoriferous substances of the flower is allowed to be absorbed by a highly volatile chemical solvent

like perfumery grade hexane, petroleum ether etc., and then the solvent is evaporated leaving behind the odoriferous principles, known as concrete. The floral concrete thus obtained contains some impurities like plant waxes, albumin, colouring matter etc. These are removed by further evaporation with absolute alcohol. The pure material thus obtained is known as absolute. Ideal Solvent properties 1. It should completely and quickly dissolve all the odoriferous principles of the flowers, yet as little as possible of such invert matters as waxes, pigments, albuminous compounds etc. In other words the solvent should be selective. 2. It should possess a sufficiently low boiling point to permit its being easily removed without resorting to higher temperatures. Yet the boiling point should not be too low, as this would involve considerable solvent loss by evaporation in the warm climate. 3. The solvent must not dissolve in water, since the water present in the flowers would dissolve and accumulate in the solvents. 4. The solvent must be chemically inert i.e. it should not react with the constituents of the flower oil. 5. The solvent should have a uniform boiling point when evaporated and should not leave any residue. 6. The solvent should be low priced and, if possible, non-flammable. Petroleum Ether: The fraction with boiling point ranges 30-70oC are commercially called petroleum ether, consisting of saturated paraffin’s, mainly pentane and hexane. Because of their chemical inertness and complete volatility, these fractions are particularly suited for the production of floral essences. A further advantage lies in their selective power of dissolving; they yield products which contain relatively little wax, albuminous and colouring matter, but correspondingly more of the odoriferous compounds. The petroleum ether must be free from sulphur and nitrogenous compounds when testing petroleum ether for use in extraction work, special attention must be paid to the presence of a non-volatile residue. For this purpose a sample of 50ml should be evaporated in a glass or porcelain dish at a temperature not exceeding 40o C. After complete evaporation, the glass dish should show no residual odour whatsoever, but especially not indicating the presence of kerosene or sulphur compounds. Although petroleum ether is the best solvent found so far for flower extraction, it possesses some inherent disadvantage. For example, due to evaporation and low boiling point of gaseous fractions, there is relatively high solvent loss in the course of extraction. It is readily

inflammable and dangerous to work with. Also, petroleum ether is quite costly and can be used only for more expensive essential oil. Benzene (Benzol): Benzene ranks next to petroleum ether as a solvent for the extraction of flowers. It is a coaltar product made by treating and purifying coaltar naphtha with sulphuric acid and subsequently with sodium hydroxide. The industrial ‘benzol’ often contains pyredine, carbondisulphide and thiophene. It has got a uniform boiling point and thus solvent losses are comparatively reduced. The boiling point is at 81 oC which is relatively high and which makes it rather difficult to remove the last traces of solvent from the concentrated flower oil production lies in its high dissolving power. It dissolves not only the odoriferous principles but also other materials such as wax, albuminous and colouring matter, so that the final flower oil extracted with benzene are dark highly viscous, often almost solid mass, which can be purified only under considerable difficulties. Compared with petroleum ether, benzene, usually gives much higher yields of concrete, due to the higher amount of inert wax, albuminous and colouring matter present. Hence, used in the production of low priced oils. Major equipment and Machinery required The major equipment and machinery required for jasmine concrete production are stainless steel extractors, condensers, solvent rectification assembly, storage tanks, chilling units, vacuum pumps, boiler etc. Extraction Process: The extractions to be used for solvent extraction may be stationary or rotary, commonly with a capacity or about 1200 litres and holding upto 200 kg of flowers per charge. Three to four washings with solvent are usually required. About five hours are required for processing a batch of flowers, including final steam distillation of the spent material to recover solvent. The concentrated washings are filtered and then evaporated in a water bath type of still in which temperature never reaches 60oC. Final purification of the oil in order to obtain the concentrated oil and to get rid of waxes or albuminous impurities may be done under vacuum to precipitate out the impurities. The Chief disadvantages of the process are the need for comparatively elaborate and expensive equipment and for precision control by trained supervisions. Hence solvent extraction will probably never replace distillation in rural areas or among the backward populations. For the low priced oils, the process is uneconomical as in this a considerable amount of purified solvent is unavoidably lost, an amount that might perhaps represent the difference between profit and loss in the enterprise.

The solvent extraction method involves the following steps: a) Dissolving the perfume material in the solvent by treating the flowers with the solvent (1:2 ratio). The solvents normally used are petroleum ether or Food grade Hexane with boiling point from 60 to 80oC. The concrete extracted from the latter solvent is much preferred in Indian and foreign perfumery markets. In order to ensure the complete extraction of the natural perfume, the container having the flowers and solvent may be rotated slowly for about 5 minutes in the rotary type or extractor b) The second step involves the removal of the solvent from the perfume material by evaporation. This is the achieved by heating of the evaporator indirectly in a water bath till about 90% of the solvent is recovered by condensation. The remaining 10% of the solvent containing all the aromatic oil and waxy material is transferred to vacuum distillation unit where the complete removal of the solvent is effected and the concrete is left in the still in the form of the molten wax. This concrete, when still hot is transferred to glass or aluminium containers for storage. 2. Enfleurage or Cold Fat Extraction • Where the distillation may have deleterious effects on an essential oil through hydrolysis, polymerization or resinification • where delicate oils become 'lost' in large volumes of water • where the flowers continue to produce fragrance after the harvest Enfleurage is usually practiced. Fat has a high absorption capacity and, if brought in contact with fragrant flowers, readily absorbs the perfume emitted. This principle methodically applied on a large scale, constitutes enfleurage. The success of enfleurage depends, to a great extent, upon the quality of the fat base (called the corps) employed. Utmost care must be exercised when preparing corps. If the corps is too hard, the blossoms will not have sufficient contact with the fat, curtailing the power of absorption and resulting in a subnormal yield of flower oil. On the other hand, if it is too soft, it may engulf the flowers, so that the exhausted ones are difficult to remove and retain adhering fat which entails considerable shrinkage and loss of corps. The consistency of the corps must, therefore, be such that it offers a semi-hard surface from which the exhausted flowers can easily be removed. Since the whole process of enfleurage is carried out in cool cellars, every manufacturer must prepare his corps according to the temperature prevailing in his cellars during the month of the flower harvest. Many years of experience have proved that a mixture of one part of highly purified tallow (ox or sheep fat) and two parts of lard (fat of swine) gives the corps of required type.

Procedure: Enfleurage and Defleurage Every enfleurage building is equipped with thousands of so-called chassis, which serve as vehicles for holding the fat corps during the process. A chassis consists of a rectangular wooden frame of 5 cm high, about 50 cm long and about 40 cm wide. The frame holds a glass plate upon both sides of which the fat corps @ of 360 g each side or 1 kg for every 2.5 kg of flowers is applied with the help of a spatula at the beginning of the enfleurage process. When piled one above the other, the chassis form airtight compartments, with a layer of fat on the upper and lower side of each glass plate. Every morning during the harvest, freshly-picked flowers arrive and having first been cleaned of impurities, such as leaves, and stalks, are then strewn by hand on the top of the fat layer of each glass plate. Blossoms wet from dew or rain must never be employed, as any trace of moisture would turn the corps rancid. The chassis are charged and left in the cellars for 24 hours or longer, depending upon the type of flowers. During this period, the flowers come in direct contact with the fat layer on which they are resting and lose their fragrance in the absorptive fat, whereas the fat layer above them absorbs only the volatile perfume given off by the flowers. After 24 hours, when the flowers have emitted most of their oil and start to wither, they should be removed from corps with the help of tweezers, which is referred as defleurage. The careful removal of the flowers is very important to avoid any objectionable odour. Although most of the exhausted flowers will fall from the fat layer on the chassis glass plate when they are struck lightly against the working table, some flower particles will still adhere and have to be removed manually. Immediately following defleurage, the chassis are recharged with fresh flowers. For this purpose, the chassis are turned over and the fat layer which, in the previous operation, formed the top is now directly charged with flowers. In the case of jasmine, the entire enfleurage process lasts about 70 days; daily, the exhausted flowers are removed and the chassis is charged with fresh ones. At the end of the harvest, the fat is relatively saturated with the flower oil and possesses the typical fragrance. The perfumed fat must then be removed from the glass plates between the chassis. For this purpose, it is scraped off with a spatula and then carefully melted and bulked in closed containers. This final product is, called as 'Pomade' and in earlier times was directly utilized without further treatment. At the end of the enfleurage, the fat corps loses about 10% of its weight because of various manipulations. In other words, the total yield of the fragrant pomade is less than the fat corps originally applied to the chassis. Most of the loss is caused by the fat adhering to the exhausted flowers when they are removed every 24 hours.

Alcoholic Extracts (Extrait) In the early days of perfumery, the fragrant pomades were used directly without further treatment. More recently, alcoholic extracts with the use of high quality alcohol have been made. In the trade, they are known as extracts. Since no heat is applied during the process of enfleurage and while washing the pomade with alcohol, the extracts contain the natural flower oil as emitted by living flowers. The only disadvantage that exists possibly, is a slight fatty 'by note' which can be eliminated to a certain extent by freezing and filtering the alcoholic washings. However, this 'by note' is not always considered objectionable, as it imports a certain roundness and fixation value to the finished perfumes, especially in conjunction with synthetic aromatics. In order to prepare extracts, the pomades are usually processed during the winter months when the factories are not busy with other work. For this purpose, the pomade is charged into specially designed equipment called 'batteuse' which is a closed copper vessel, heavily tinned inside and equipped with strong stirrers around a vertical shaft. Several batteuses are arranged in batteries, the stirrers of each battery being driven by a powerful motor. This work, which goes on for several months, is carried out in cool cellars in order to prevent loss of alcohol by evaporation. The alcohol used in the process travels from one batch of pomade to the next (constituting in turn the third, second and first washings of successive batches), until it becomes enriched with flower oil and is drawn out as the alcoholic extra it. For the last washing, fresh alcohol is used which also, in turn, becomes gradually enriched by the continuous process just described. When extended to a further or fifth washing, this method extracts the pomades so efficiently that the exhausted fat is quite odorless. Being useless for re-use in enfleurage, it is usually employed for making soap. The fully-saturated washing is run through a refrigerator and cooled to well below freezing temperature. If possible, to -15°C. Most of the fat dissolved in the alcohol separates by this treatment. The cold alcoholic solution is then filtered at a low temperature. The quantity of alcohol to be used for washing each batch for pomade is calculated with a view to finally obtain 1 kg of extract per kg of pomade. Obviously, some alcohol is lost by evaporation during the process of stirring. 3. Maceration or Hot Fat Extraction Certain flowers such as jasmine and tuberose give their greatest yield of floweroil upon extraction with enfleurage, because their physiological activities continue for a further 24 hours or more after harvesting. However, the physiological activities in flowers like the rose, orange, violet and acacia are stopped after picking; therefore, when

extracted or distilled they yield only as much oil as contained in the flowers at that moment. Since no further oil develops in these flowers, the long and rather complicated method of enfleurage would prove ineffective. Hence, other methods must be resorted to whereby a medium actually penetrates the plant tissue and dissolves all the flower-oil present in the oil glands. In this case, a batch of hot fat is systematically treated with several batches of flowers until it becomes quite saturated with the flower perfume. The fragrant fat thus obtained, depending upon the flowers used, is called 'orange pomade' or 'rose pomade', and is sold as such or it may be treated further by washing it with strong alcohol, exactly as jasmine and tuberose pomades are obtained by enfleurage. Every extraction lasts for about one-and-a-half hours. On a commercial scale, a batch of 80 kg corps is heated to about 80°C temperature and charged with 20 kg of fresh flowers each time, until 1 kg of corps has been treated with about 2 to 2.5 kg of flowers. During the process of maceration, when the corps is left standing for about an hour, it cools and solidifies. The mass is then reheated, melted and strained through metal sieves and filter bags, whereby the exhausted flowers arc eliminated. The method of maceration is rather cumbersome, but it served its purpose in the old days when no better process was available. Its products (extraits and absolutes of maceration) often show a fatty by-note which originates from the fat corps and modifies the character of the original flower perfume. A further disadvantage is that the fat content in the absolute of maceration easily turns rancid, thereby developing a sharp disagreeable note. 4. Expression The expression of essential oil is practiced in some special cases, as in the production of citrus oils from the juices and waste rinds from citrus canning factories. In general, expression involves squeezing any plant material at great pressures in order to press out the oils or other liquids. The process is carried out by hand-operated presses or crushers in isolated rural areas, or by gigantic mechanical presses in industrial centers. In the production of citrus juices, the oil is unavoidably expressed from the fruit. The oil is then separated from the juice by centrifuging.

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