CHAPTER-1 INTRODUCTION

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Humankind has used plants for healing for many thousands of years, and it's from this tradition of that the use of aromatic plant compounds is medicine began. Oils were used in the embalming process, in medicine andin purification rituals. Frankincense, Myrrh, Galbanum, Cinnamon, Cassia, Rosemary, Hyssop and Spikenard are noted for being used for anointing rituals and healing of the sick. With the continual bombardment of viral, bacterial, parasitic and fungal contamination in our world, essential oils are a great benefit to help protect our bodiesand homes from this onslaught of pathogens. Immune system need support and essential oils can give it.

Because of the enormous amount of raw product used to make wholly natural essential oils, lots of products on the market have been polluted with lower quality, commercial – grade oils or contain other chemical substances to reduce the cost or increase the profit margin – afact not usually revealed on the label. This is why it is important to study the chemical composition of the volatile fraction once the essential oil is extracted. This fraction is characterized by the complexity in the separation of its components, which belong to various classes of compounds and which are present in a wide range of concentrations.

Therefore it is complicated to establish a composition profile of essential oils. The gas chromatographic method (GC) is almost exclusively used for the qualitative analysis of volatiles. The analysisof essential oils was developed in parallel with the technological developments in GC, such as stationary phases, detection devices, etc. However, advances in instrumentation were not the only important factor in the development of analytical methods for essential oils in plants. Sample extraction and concentration were also improved. The most outstanding improvements in the determination of the composition of

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essential oils came from the introduction of tandem techniques involvingprior/further chromatography or spectroscopy.

Analysis of Essential Oil is done by using Gas Chromatography with Mass Spectrometer. The qualitative and quantitative analysis is done to know the constituents in the oil and the percentage of components present in the oil respectively, by doing so we can know the purity of that particular oil.

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CHAPTER -2

LITERATURESURVEY

2.1. WHAT ARE ESSENTIAL OILS? Essential oils are concentrated volatile aromatic compounds produced

by plants - the easily evaporated essences that give plants theirwonderful scents. Each of these complex precious liquids is extractedfrom a particular species of plant life. Each plant species originates

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in certain regions of the world, with particular environmentalconditions and neighboring fauna and flora.

Essential oils are frequently referred to as the “life force” of plants.Unlike fatty oils, these "essential" oils are volatile, highly concentrated, substances extracted from flowers, leaves, stems, roots, seeds, bark, resin or fruit rinds. The amount of essential oils found inthese plants can be anywhere from 0.01 percent to 10 percent of the total. That's why tons of plant material are required for just a few hundred pounds of oil. These oils have potent antimicrobial factors, having wide range of therapeutic constituents. These oils are often usedfor their flavor and their therapeutic or odoriferous properties, in a wide selection of products such as foods, medicines, and cosmetics. Beware of imitations. Essential oils cannot be substituted with synthetics. Only pure oils contain a full spectrum of compounds that cheap imitations simply cannot duplicate.

2.2. OVERVIEW OF MENTHA OIL

India is world’s largest producer and exporter of mint oil. Mint oil andits constituents and derivatives are used in food, pharmaceutical and perfumery and flavouring industry. Its main constituent, menthol, is used in the manufacture of lozenges, toothpastes, pain balms, cold balms, Dabur Pudin Hara, etc. The basic raw material for mint oil is leaves of a plant Mentha arvensis. The oil is used for treating certain stomach disorders like indigestion, gas problem, acidity, etc. It is themain ingredient of ayurvedic medicines like Daburs ‘Pudin Hara’. The oilis a natural source of menthol, which is the main ingredient of cough drops and ointments like Vicks Vaporub, etc.

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fig1) mentha tree

Composition of Oil: The natural oil yields on an average 40-50% mentholand 50-60% dementholised oil, which can be used both in confectioneryand medicine in place of imported peppermint oil. Japanese mint oil isnot distinguished from the peppermint oil in the Indian trade. Thedementholised oil has been found to contain menthyl acetate (24.4%),free menthol (44.8%), menthone (24.6%) and hydrocarbons (6.2%). Amongthe hydrocarbons, alpha-pinene, a-1-limonene, carophyllene and cademeneare present.

Cultivation Mentha arvensis is cultivated in India in the semi- temperate regions in thefoothills of Himalayas in Punjab, Himachal Pradesh, Uttar Pradesh andBihar. In 1997 the area under mentha in U.P. went up to 40,000 hectaresfrom 20,000 hectares in 1996 because some of the sugarcane farmers tookup its cultivation in view of non-payment of arrears by the sugar millsand closure of several mill.

Global ScenarioThe native Place of Mentha is Japan. After Japan, its cultivation began in Argentina,Brazil and China. Then it started in India. On global front, India remains in top position in terms of Mentha oil production and exports are concerned. Out of total Mentha oil produced, 75% is contributed from Mentha arvensis (mainly to produce menthol), 18% is peppermint and 7% is spearmint. Out of total M. arvensis oil produced: India contributes 73%, China 18% and others 9%.About 30-40% of total

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Mentha production is consumed by India, followed by China, US and European countries.

Indian scenarioThe large scale commercial cultivation of Mentha is done in UP (Bilaspur, Rampur , Chandausi, Sambhal, Barabanki , Bareilly , Sitapur etc.) , Punjab (Jalandhar), Haryana (Ambala) , Himachal and Bihar (Muzuffarpur).India produces about 27,600 tons of mentha crude oil per annum (avg. forlast five years). The output is increasing in recent years and a record production close to 35,000 tons was seen in 2009.

Share in mentha oil distribution

Source: Spices Board Of India Mentha oil production trend in india

Source : spices board of india

Mentha oil export

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Source: spices board of india

Mentha oil export from india

Source : spices board of indiaMentha oil price seasonality

Source: spices board of india

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Uses of mint oil:-Physiological actionsAnesthetic, Antiphlogistic, Antidepressant, Anti-microbal, Antiseptic, Anti-spasmodic, Carminative, Digestive, Expectorant, Nervine, Stomachic, TonicOther Uses:Not recommended for homeopathic treatment. Benefits the nervous system and useful in treatment of stress-related conditions such as headache, insomnia and nervous tension. Useful in treatment of skin problems such as acne, boils and ulcers. Useful in the treatment of circulation, muscles and joints complications and relieves arthritis, inflamed joints, muscular pains, rheumatism and sprains. Benefits the immune systems and useful in the treatment of colds, flu and infections. Usefulwith respiratory infections such as asthma, throat infections, laryngitis and fever. Useful in the treatment of the genitourinary system disorders such as amenorrhoea, labor pain and dysmenorrhoea.Industrial Uses:Extensively used as fragrance component in soaps, detergents, cosmetics and perfumes, toothpastes, and industrial fragrances. Extensively used as flavoring agent in food products such as confectionery, liquors, and chewing gums. Used in cough syrups, lozenges and herbal teas in the formof menthol.aSafety Data:Non-toxic. Non-irritant. Not to be consumed internally. Avoid during pregnancy. Use with care. Keep out of the reach of children.

2.3 OVERVIEW OF PALMAROSA OIL :- Palmarosa oil has a sweet floral, with a hint of rose smell and is pale yellow in color with a nearly watery viscosity.Area under cultivation It is cultivated in Uttar Pradesh, Madhya Pradesh, Jodhpur (Rajasthan),Karnataka, Maharashtra and Tamil Nadu. Family: Poaceae Plant Description Palmarosa is a wild growing plant related to lemongrass. It has fragrantleaves, long slender stems and terminal flowering tops. It is a droughthardy grass attaining a height of 1.5 to 2.5m having hairy and fibrousshallow root system with long linear lanceolate leaves. It produces

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large fawn coloured inflorescence containing while, hairy star likespiked flowers. Centre of Origin: India

fig7) palmarosa

Soil

A well-drained sandy loam soil with soil pH of 7.5-8.5 is ideal forcultivation of Palmarosa and receiving rainfall of about 150 cm annuallyis an It can also be cultivated in well-drained clay loam soil which arefree from water logging.

ClimateWarm tropical climate upto 300m elevation in the foothills is suitable for cultivation of Palmarosa. Temperature ranging from 10 – 360oC with annual rainfall around 1000mm and ample sunshine are congenial for its growth. Moist and warm climate throughout the year accelerates its growth. Areas, which are affected by severe frost, are not suitable as the frost kills the grass and reduces the oil content.

Manuring & Fertilization Palmarosa is a long duration crop and removes substantial quantities of nutrients from the soil for producing herbage. Therefore, use of FYM @ 10 t /ha, 40 kg N, 50 kg P and 40 kg K as a 2 5 2 basal dose is recommended. About 60kg N/ha is applied in three split doses during the growing season. The application of NPK should be repeated in subsequent years. In fertile soils, manuring may not be required for the first two years. By manuring rich soils, the vegetative growth is increased and oil content may be slightly reduced.

Harvesting The essential oil is distributed in all parts of the grass, viz., flower

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heads, leaves and stems, the flower heads containing the major portion. It is recommended to harvest the crop 7-10 days after opening of flowers.

Uses Oil of Palmarosa is used in perfumery, particularly for flavoring tobacco and for blending of soaps due to the lasting rose-note it imparts to the blend. It also serves as a source for very high grade geraniol. Geraniol is highly valued as a perfume and as a starting material for large chemicals, viz., geranyl esters that have a permanentrose-like odour.

The therapeutic properties of Palmarosa oil are antiseptic, antiviral, bactericide, cytophylactic, digestive, febrifuge and hydrating.Palmarosaoil calms the mind, yet has an uplifting effect, while clearing muddled thinking.

It is used to counter physical and nervous exhaustion, stress-related problems and nervousness.It is most useful during convalescence and cools the body of fever, while aiding the digestive system, helping to clear intestinal infection, digestive atonia and anorexia nervosa. It iseffective in relieving sore, stiff muscles.

Palmarosa oil moisturizes the skin, while balancing the hydration levelsand stimulating cell regeneration. It balances production of sebum, to keep the skin supple and elastic and is valuable for use with acne, dermatitis, preventing scarring, rejuvenating and regenerating the skin,as well as fighting minor skin infections, sore tired feet and athlete'sfoot.

Distillation The grass is either distilled afresh or is allowed to wilt for 24 hours.Wilting reduces the moisture content and allows a larger quantity of grass to be packed into the still, thus economizing the fuel use. For good quality oil, it is advisable to adopt steam-distillation. The equipment for distillation consists of a boiler to produce steam, a distillation tub, a condenser and one to three separators. The distillation tub is made of mild steel and has a perforated bottom over which the grass rests. The tub has a steam inlet pipe at the bottom. A removable lid is fitted on to the top. Charging and discharging can be done in perforated cages with iron chains, which can be lowered in the tub with the help of a chain- pulley block. Different types of condensers are available, but tubular condensers are better than others.

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The condenser is provided with an inlet and outlet by means of which cold water is made to flow through the chamber to cool the pipes when the distillate flows through them.

The constituents of palmarosa oil are:- The main chemical components ofpalmarosa oil are myrcene, linalool, geraniol, geranyl acetate,dipentene and limonene.

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2.4) AN INTRODUCTION TO CHROMATOGRAPHIC METHODS

Chromatography was invented by the Russian botanist Mikhail Tswett shortly after the turn of this century. He passed solutions containing plant pigments, such as chlorophylls and xanthophylls, through glass columns packed with finely divided calcium carbonate. The separated species appeared as colored bands on the column, which accounts for the name he choose for the method (Greek: chroma meaning "color" and graphein meaning "to write ").

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Chromatography is a technique in which the components of a mixture are separated based on the rates at which they are carried through a stationary phase by a gaseous or liquid mobile phase.

The technique, as described by Tswett was largely ignored for a along time and it was not until the late 1930s and early 1940s that Martin andSynge introduced liquid-liquid chromatography by supporting the stationary phase, in this case water, on silica in a packed bed and usedit to separate some acetyl amino acids. In their paper, they recommendedreplacing the liquid mobile phase by a suitable gas, as the transfer of sample between the two phases would be faster, and thus provide more efficient separations.

In this manner, the concept of gas chromatography was created. Chromatographic methods are of two types. In column chromatography, the stationary phase is held in a narrow tube and the mobile phase is forcedthrough the tube under pressure or by gravity. In planar chromatography, the stationary phase is supported on a flat plate or in the pores of a paper. Here the mobile phase moves through the stationary phase by capillary action or under the influence of gravity.

Liquid chromatography can be performed in columns and on planar surfaces, but gas

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chromatography is restricted to column procedures.TABLE1Mobile phase Stationary phaseGas Liquid (gas-liquid

chromatography )Gas Soild (gas-solid

chromatography)Liquid Liquid (liquid-liquid

chromatography)Liquid Solid (liquid-liquid

chromatography)

Fig. column chromatography

Fig.planar chromatography

Today, chromatography is an extremely versatile technique; it can separate gases, and volatile substances by GC, nonvolatile chemicals andmaterials of extremely high molecular weight (including biopolymers) by LC and if necessary very inexpensively by TLC. All three techniques,

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(GC), (LC) and TLC have common features that classify them as chromatography systems.

Chromatography is a separation process that is achieved by distributing the components of a mixture between two phases, a stationary phase and amobile phase. Those components held preferentially in the stationary phase are retained longer in the system than those that are distributed selectively in the mobile phase. As a consequence, solutes are eluted from the system as local concentrations in the mobile phase in the orderof their increasing distribution coefficients with respect tothe stationary phase.

Chromatogram is a plot of some function of solute concentration versus elution time or elution volume.

Paper chromatography:-In paper chromatography, the stationary phase is a specially manufactured porous paper. The samples are added to one end of the sheetof paper and dipped into the liquid or mobile phase. The solvent is drawn through the paper by capillary action and the molecules are distributed by partition between the mobile and stationary phase. The partition coefficient, k, similar to the distribution coefficient for extraction, is the equilibrium constant for the distribution of molecules between the mobile phase and the stationary phase. It is this equilibrium that separates the components. Different inks and dyes, depending on their molecular structures and interactions with the paper and mobile phase, will adhere to the paper more or less than the other compounds allowing a quick and efficient separation.

Adsorption Chromatography:-

Adsorption – of solute on surface of stationary phase; for polar non-ionic compounds.

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Ion Exchange Chromatography – attraction of ions of opposite charge; forionic compounds anions or cations.

Partition chromatography - based on the relative solubility of analyte in mobile and stationary phases.

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Partition coefficient, KD , based on thermodynamic equilibrium ratio of analyteCs concentration in stationary phaseCm concentration in mobile phaseKD = CS/ Cm

The same principle as Liquid Liquid Extraction.

Size Exclusion chromatography (gel filtration, gel permeation) – separates molecules by size; sieving - not real interaction, small molecules travel longer.

Affinity chromatography – specific interactions like a particular antibody to protein.

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Gas chromatography (GC)This technique uses a gas as the mobile phase, and the stationary phase can either be a solid or a non-volatile liquid (in which case small inert particles such as diatomaceous earth are coated with the liquid so that a large surface area exists for the solute to equilibrate with). If a solid stationary phase is used the technique is described as gas-solid adsorption chromatography, and if the stationary phase is liquid it is called gas-liquid partition chromatography.

Liquid chromatography (LC)Liquid chromatography is similar to gas chromatography but uses a liquidinstead of a gaseous mobile phase. The stationary phase is usually an inert solid such as silica gel (SiO2.xH2O), alumina (Al2O3 .xH2O) or cellulose supported in a glass column. The adsorbing properties of silica and alumina are reduced if they absorb water, but the reduction is reversed by heating to 200–400 °C. Silica is slightly acidic, and

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readily adsorbs basic solutes. On the other hand, alumina is slightly basic and strongly adsorbs acidic solutes. Other stationary phases that can be used include magnesia, MgO.xH2O (good for separating unsaturated organic compounds); and dextran (a polymer of glucose) cross-linked withpropan-1,2,3-triol (glycerol,CH2OHCHOHCH2OH), which is sold as Sephadex and can separate compounds such as purines.

A wide range of solvents are used in this technique, including hydrocarbons, aromatic compounds, alcohols, ketones, halogen compounds and esters. A mixture of solvents can also be used. The optimum solvent is chosen by running experiments on a small scale using TLC plates.

Thin layer chromatography:-Thin-layer chromatography or TLC, is a solid-liquid form of chromatography where the stationary phase is normally a polar absorbent and the mobile phase can be a single solvent or combination of solvents.TLC is a quick, inexpensive microscale technique that can be used to:

• determine the number of components in a mixture• verify a substance’s identity• monitor the progress of a reaction• determine appropriate conditions for column chromatography• analyze the fractions obtained from column chromatography.

In thin-layer chromatography, the stationary phase is a polar absorbent,usually finely ground alumina or silica particles. This absorbent is coated on a glass slide or plastic sheet creating a thin layer of the particular stationary phase. Almost all mixtures of solvents can be usedas the mobile phase. By manipulating the mobile phase, organic compoundscan be separated.

Mixture of A & B free in mobile phase and absorbed on the stationary phase.

The equilibrium between the free and absorbed states depends on three factors:

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• the polarity and size of the molecule• the polarity of the stationary phase• the polarity of the solvent

Dynamic equilibrium between A & B and the mobile and stationary phase.

In TLC, the stationary phase is typically alumina (Al2O3 .xH2O)n or silica gel (SiO2.xH2O)n .The covalent network of these absorbents create very polar materials.TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLCand it takes little time for an analysis (about 5-10 minutes).

TLC consists of three steps - spotting, development, and visualization. First the sample to be analyzed is dissolved in a volatile (easily evaporated) solvent to produce a very dilute (about 1%) solution.

Spotting consists of using a micropipet to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin layer of powdered silica gel that has been coated onto a plastic sheet. The spotting solvent quickly evaporates and leaves behind a small spot of the material.

Development consists of placing the bottom of the TLC plate into a shallow pool of a development solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it moves over the original spot. A competition is set up between the silica gel plate and the development solvent for the spotted material. The very polar silica gel tries to hold the spot in its original place and the solvent tries to move the spot along with it as it travels up the plate. The outcome depends upon a balance among three polarities - that of the plate, the development solvent and the spot material. If the developmentsolvent is polar enough, the spot will move some distance from its original location. Different components in the original spot, having

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different polarities, will move different distances from the original spot location and show up as separate spots. When the solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked with a pencil, and the solvent allowed to evaporate.

Visualization of colored compounds is simple – the spots can be directlyobserved afterdevelopment . Because most compounds are colorless however, a visualization method is needed .The silica gel on the TLC plate is impregnated with a fluorescent material that glows under ultraviolet (UV) light. A spot will interfere with the fluorescence and appear as a dark spot on a glowing background. While under the UV light, the spots can be outlined with a pencil to mark their locations. A second method of visualization is accomplished by placing the plate into iodine vaporsfor a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is good practice to use at least two visualization techniques in case a compound does not show up with one particular method.

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The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the distance traveled by the substance divided by the distance traveled by the solvent. Its value is always between zero and one. The Rf value is the “retardation factor” or the “ratio-to-front”value expressed as a decimal fraction.

Rf = Y/X (always ≤ 1)

Chromatography nomenclature

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The baseline is any part of the chromatogram where only mobile phase is emerging from the column.

The peak maximum is the highest point of the peak.

The injection point is that point in time/position time when/where the sample is placed on the column.

The dead point is the position of the peak-maximum of an unretained solute.

The dead time (to) is the time elapsed between the injection point and thedead point.

The dead volume (Vo) is the volume of mobile phase passed through the column between the injection point and the dead point. Thus, Vo = Qto where Q is the flow rate in ml/min.

The retention time (tr) is the time elapsed between the injection point and the peak maximum. Each solute has a characteristic retention time.

The retention volume (Vr) is the volume of mobile phase passed through the column between the injection point and the peak maximum.Thus, Vr = Qtr where Q is the flow rate in ml/min.

Each solute will also have a characteristic retention volume.

The corrected retention time (t'r) is the time elapsed between the dead point and the peak maximum.

The corrected retention volume (V'r) is the volume of mobile phase passedthrough the column between the dead point and the peak maximum.It will also be the retention volume minus the dead volume.

Thus, V'r = Vr – Vo = Q(tr – to) where Q is the flow rate in ml/min.

The peak height (h) is the distance between the peak maximum and the baseline geometrically produced beneath the peak.

Factors Controlling Retention

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Vr = Vm + KVS

or V'r = K VS

where,(Vm) is the volume of mobile phase in the column(VS) is the volume of stationary phase in the column,(K) is the distribution coefficient of the solute between the phases,and (V'r) is the corrected retention volume i.e., ( Vr – Vm)

2.5) INTRODUCTION TO MASS SPECTROSCOPY

Mass Spectrometry is a powerful technique for identifying unknowns, studying molecular structure, and probing the fundamental principles of chemistry. Applications of mass spectrometry include identifying and quantifying pesticides in water samples, in identifying steroids in athletes, determining metals at ppq (Parts Per Quadrillion) levels in water samples,carbon-14 dating the Shroud of Turin using only 40 mg of sample (1), looking for life on Mars, determining the mass of an 28Si atom with an accuracy of 70 ppt(2), and studying the effect of molecularcollision angle on reaction mechanisms.

Mass spectrometry is essentially a technique for "weighing" molecules.*

Obviously, this is not done with a conventional balance or scale. Instead, mass spectrometry is based upon themotion of a charged particle, called an ion, in an electric or magnetic field. The mass to charge ratio (m/z)** of the ion effects this motion. Since the charge of an electron is known, the mass to charge ratio a measurement of an ion'smass.

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MASS SPECTROMETER

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An instrument which measures the ratio of mass to the number of charges of ions producedfrom elements and compounds. It is also of value in performing fundamental studies of theproperties of gaseous ions. A mass spectrometer is similar to a prism. In the prism, light isseparated into its component wavelengths which are then detected with anoptical receptor,such as visualization. Similarly, in a mass spectrometer the generated ions are separated inthe mass analyzer, digitized and detected by an ion detector.

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The basic diagram of a spectrometer.

Understanding Mass SpectrometryTo understand the basic principles of mass spectrometry, consider a person standing at the top of a tower on a windy day. The person picks up various balls and drops them, one by one,from the tower. As each ballfalls, wind deflects it along a curved path. The masses of the balls affect how they fall. A bowling ball, for example, is much heavier than a basketball andis therefore harder to move. As a result, a bowling ballfollows a different path than a baseball.In a mass spectrometer, the same thing is happening, except it's atoms and molecules that arebeing deflected, and it's electric or magnetic fields causing the deflection.

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Sample Introduction TechniquesIn order to perform mass analysis on a sample, which is initially at atmospheric pressure (760 mmHg), it must be introduced into the instrument in such a way that the vacuum inside the instrument remains relatively unchanged (~10-6 torr). The most common methods of sample introduction are direct insertion with a probe or plate commonly used with MALDI-MS, direct infusion or injection into the ionization source such as ESI-MS.

Direct Insertion:Using an insertion probe/plate is a very simple way to introduce a sample into an instrument.The sample is first placed onto a probe and then inserted into the ionization region of the mass spectrometer, typically through a vacuum interlock. The sample is then subjected to any number of desorption processes, such as laser desorption or direct heating, to facilitate vaporization and ionization.

Direct Infusion:A simple capillary or a capillary column is used to introduce a sample as a gas or in solution.Direct infusion is also useful because it can efficiently introduce small quantities of sampleinto a mass spectrometer without compromising the vacuum. Capillary columns are routinelyused to interface separation techniques with the ionization source of a mass spectrometer.These techniques, including gas chromatography (GC) and liquid chromatography (LC), alsoserve to separate a solution’s different components prior to mass analysis. In gaschromatography, separation of different components occurs within a glasscapillary column.

As the vaporized sample exits the gas chromatograph, it is directly introduced into the massSpectrometer.

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IONIZATION TECHNIQUES:Electron Ionization. Electron Ionization (EI) is the most common ionization technique used for mass spectrometry. EI works well for many gas phase molecules, but it does have some limitations. Although the mass spectra are very reproducible and are widely used for spectral libraries, EI causes extensive fragmentation so that the molecular ion is not observed for many compounds. Fragmentation is useful because it provides structural information for interpreting unknown spectra.

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Advantages Widely used technique when coupled to GC Suitable for volatile organic compounds

– eg hydrocarbons, oils, flavours, fragrances Not really coupled to LC today Also called electron impact Produces M+. radical cation giving molecular weight Produces abundant fragment ions

Chemical Ionization:- Chemical Ionization (CI) is a _soft” ionization technique that produces ions with little excess energy. As a result, less fragmentation is observed in the mass spectrum. Since this increases the abundance of the molecular ion, the technique is complimentary to 70 eV EI. CI is often used to verify the molecular massof an unknown. Only slight modifications of an EI source region are required for CI experiments.

Reagent (R) + e- → R+ + 2 e-R+ + RH → RH+ + RRH+ + Analyte (A) → AH+ + R

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Negative chemical ionization (NCI) typically requires an analyte that contains electron capturing moieties (e.g., fluorine atoms or nitrobenzyl groups). Such moieties significantly increase the sensitivity of NICI, in some cases 100 to 1000 times greater than that of electron ionization (EI).NCI is probably one of the most sensitive techniques and is used for a widevariety of small molecules with the caveat that the molecules are often chemically modified with an electron-capturing moiety prior to analysis.

While most compounds will not produce negative ions using EI or CI, many important compounds can produce negative ions and, in some cases, negative EI or CI mass spectrometry is more sensitive and selective than positive ion analysis. In fact, compounds like steroids are modified to enhance NCI.

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Advantages Produces M+H+ ions or M - H-ions Gives molecular weight Softer ionization technique

Fast Atom Bombardment and Secondary Ion Mass Spectrometry:- Fast Atom Bombardment (FAB) and Secondary Ion Mass Spectrometry (SIMS) both use high energy atoms to sputter and ionize the sample in a single step. In these techniques, a beam of rare gas neutrals (FAB) or ions (SIMS) is focusedon the liquid or solid sample. The impact of this high energy beam causes the analyte molecules to sputter into the gas phase and ionize ina single step. The exact mechanism of this process is not well understood, but these techniques work well for compounds with molecular weights up to a few thousand dalton. Since no heating is required, sputtering techniques (especially FAB) are useful for studying thermallylabile compounds that decompose in conventional inlets.

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FAB matrix -- Facilitating the desorption and ionization process, the FAB matrix is a non-volatile liquid material that serves to constantly replenish the surface with new sample as it is bombarded by the incidention beam. By absorbing most of the incident energy, the matrix also minimizes sample degradation from the high-energy particle beam.

Two of the most common matrices used with FAB are m-nitrobenzyl alcohol andglycerol.

Thermal ionizationThermal ionization is based upon the generation of atomic or molecular ions at the surface ofan electrically heated filament. Samples are deposited on specially treated filaments (usuallyrhenium or tantalum), then carefully dried. The filaments are heated slowly, leading toevaporation and vaporization of the sample. It is useful for determiningthe elements thatevaporate at low temperature but require high ionization temperatures (Ca, for example). TIis generally used for precise and accurate measurement of stable isotoperatio of inorganicelements. It is also used to quantify toxic trace elements in foods.

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Matrix Assisted Laser Desorption/Ionization.Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)was first introduced in 1988 by Tanaka, Karas, and Hillenkamp. It has since become a widespread analytical tool for peptides, proteins, and most other biomolecules (oligonucleotides, carbohydrates, natural products, and lipids).

Matrix Assisted Laser Desorption/Ionization (MALDI) is used to analyze extremely large molecules. This technique directly ionizes and vaporizesthe analyte from the condensed phase. MALDI is often used for the analysis of synthetic and natural polymers, proteins, and peptides. Analysis of compounds with molecular weights up to 200,000 dalton is possible and this high mass limit is continually increasing.

In MALDI, both desorption and ionization are induced by a single laser pulse.The sample is prepared by mixing the analyte and a matrix compoundchosen to absorb the laser wavelength. This is placed on a probe tip anddried. A vacuum lock is used to insert the probe into the source region

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of the mass spectrometer. A laser beam is then focused on this dried mixture and the energy from a laser pulse is absorbed by the matrix. This energy ejects analyte ions from the surface so that a mass spectrumis acquired for each laser pulse.

MALDI matrix -- A nonvolatile solid material facilitates the desorption and ionization process by absorbing the laser radiation. As a result, both the matrix and any sample embedded in the matrix are vaporized. Thematrix also serves to minimize sample damage from laser radiation by absorbing most of the incident energy.

Advantages practical mass range of up to 300,000 Da. Species of much greater masshave been observed using a high current detector; typical sensitivity on the order of low femtomole to low picomole. Attomole sensitivity is possible; soft ionization with little to no fragmentation observed; tolerance of salts in millimolar concentrations; suitable for the analysis of complex mixtures.

Disadvantages Matrix background, which can be a problem for compounds below a mass of 700 Da. This background interferences is highly dependent on the matrix material; possibility of photo-degradation by laser desorption/ionization;

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Acidic matrix used in MALDI my cause degradation on some compounds.

Electrospray IonizationElectrospray ionization (ESI) is a method routinely used with peptides, proteins, carbohydrates, small oligonucleotides, synthetic polymers, andlipids. ESI produces gaseous ionized molecules directly from a liquid solution. It operates by creating a fine spray of highly charged droplets in the presence of an electric field.

Advantages practical mass range of up to 70,000 Da good sensitivity with femtomole to low picomole sensitivity typical softest ionization method, capable of generating noncovalent complexesin the gas phase easily adaptable to liquid chromatography easily adaptable to tandem mass analyzers such as ion traps and triplequadrupole instruments multiple charging allows for analysis of high mass ions with a relatively low m/z range instrument no matrix interference

Disadvantages the presence of salts and ion-pairing agents like TFA can reduce sensitivity complex mixtures can reduce sensitivity simultaneous mixture analysis can be poor

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multiple charging can be confusing especially in mixture analysis sample purity is important carryover from sample to sample

Nanoelectrospray Ionization (NanoESI)Low flow electrospray, originally described by Wilm and Mann, has been callednanoelectrospray, nanospray, and micro-electrospray. This ionization source is a variation onESI, where the spray needle has been made very small and is positioned close to the entranceto the mass analyzer. The end result of this rather simple adjustment isincreased efficiency,which includes a reduction in the amount of sample needed.

The flow rates for nanoESI sources are on the order of tens to hundreds of nanoliters perminute. In order to obtain these low flow rates, nanoESI uses emitters of pulled and insome cases metallized glass or fused silica that have a small orifice (~5μ). Theapproximate size of droplet in nanoESI is 0.2 micron diameter which is very small as

~ 38 ~

compared to normal ESI with droplet size 1 micron diameter.

Advantages Very sensitive very low flow rates applicable to LC/MS has reasonable salt tolerance (low millimolar) multiple charging useful reasonable tolerance of mixtures Soft ionization (little fragmentation observed).Disadvantages low flow rates require specialized systems significant suppression can occur with mixtures

Atmospheric Pressure Chemical IonizationAPCI has also become an important ionization source because it generatesions directly fromsolution and it is capable of analyzing relatively nonpolar compounds. Similar toelectrospray, the liquid effluent of APCI is introduced directly into the ionization source.However, the similarity stops there. The droplets are not charged and the APCI sourcecontains a heated vaporizer, which facilitates rapid desolvation/vaporization of the droplets.Vaporized sample molecules are carried through an ion-molecule reaction region at

~ 39 ~

atmospheric pressure.

Advantages As the solvent ions are present at atmospheric pressure conditions, chemical ionization of analyte molecules is very efficient. At atmospheric pressure analyte molecules collide with the reagent ions frequently. Proton transfer (for protonation MH+ reactions) occurs in the positivemode electron transfer or proton loss, ([M-H]-) in the negative mode. Multiple charging is typically not observed presumably because the ionization process is more energetic than ESI.

Atmospheric Pressure Photoionization

Atmospheric pressure photoionization (APPI) has recently become animportant ionization source because it generates ions directly fromsolution with relatively low background and is capable of analyzingrelatively nonpolar compounds. Similar to APCI, the liquid effluent ofAPPI is introduced directly into the ionization source. The primarydifference between APCI and APPI is that the APPI vaporized samplepasses through ultra-violet light (a typical krypton light source emitsat 10.0 eV and 10.6 eV). Often, APPI is much more sensitive than ESI orAPCI and has been shown to have higher signal-to-noise ratios because oflower background ionization. Lower background signal is largely due tohigh ionization potential of standard solvents such as methanol and

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water (IP 10.85 and 12.62 eV, respectively) which are not ionized by thekrypton lamp.

In APPI Protonation, Deprotonation, Cationization reaction takes place.

Disadvantages

It can generate background ions from solvents.It requires vaporization temperatures ranging from 350-500° C, which cancause thermal degradation.

Mass AnalyzersOnce ions have been formed and introduced into the vacuum, they are subjected to electrical(DC and/or RF) or magnetic fields. Their motion under these conditions is a function of manyparameters but all include the mass-to-charge ratio. Ions can be ejectedfrom the analyser onem/z at a time or can be detected and measured, while trapped in the analyser.

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Analyzers are typically described as either continuous or pulsed. Continuous analyzers include quadrupole filters and magnetic sectors. These analyzers are similar to a filter or monochromator used for optical spectroscopy. They transmit a single selected m/z to the detectorand the mass spectrum is obtained by scanning the analyzer so that different mass to charge ratio ions are detected.Pulsed analyzers include time-of-flight, ion cyclotron resonance, and quadrupole ion trapmass spectrometers.

Mass analyzer should have following properties:-AccuracyThe accuracy of a mass measurement or concentration from a quantitative determination is a measure of how close the value obtained is to the true value. The accuracy varies dramatically from analyzer to analyzer depending on the analyzer type and resolution.Mass RangeThe range over which a mass spectrometer analyzer can operate. Quadrupole, Paul and linear ion traps tend to be limited to upper m/z values around 4000. Penning FTICR and TOF analyzers have mass limits that can extend this to well over 200,000 but there is a resolving powertrade-off at high m/z values.Resolution or resolving powerResolution is a measure of the ability of the mass spectrometer analyzerto separate two ions of different, but defined, m/z value. In simple words we can say that resolution is a measure of how well a mass spectrometer

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separates ions of different mass.Low resolution - capable of distinguishing among ions of different nominal mass that is ionsthat differ by at least one or more mass units.High resolution - capable of distinguishing among ions that differ in mass by as little as 0.0001 mass units. The resolving power A is definedas:

– To resolve two mass e.g. 950 and 951 then a resolving power A needs tobe 950 Low resolution spectrometers A = 1-2000 High resolution spectrometers A >100000

Scan SpeedAnalyzers are scanned with a regular cycle time from low to high m/z or vice versa. Quadrupole analyzers tend to be scanned linearly in mass while a magnetic analyzer is scanned exponentially to provide peaks of equal width throughout the mass range. Fast scan speeds are needed when a mass spectrometer is linked to a fast chromatographic system and TOF analyzers are currently among the best for this.

Quadrupole27. The quadrupole mass spectrometer is the most common mass analyzer. Its compact size, fast scan rate, high transmission efficiency,* and modest vacuum requirements are ideal for small inexpensive instruments. Most quadrupole instruments are limited to unitm/z resolution** and have a mass range of m/z 1000. Many benchtop instruments have a mass range of m/z 500 but research instruments are available with mass range up to m/z 4000.

Magnetic Sector. The first mass spectrometer, built by J.J. Thompson in 1897, used a magnet to measure the m/z value of an electron. M agnetic sector instruments have evolved from this same concept. Sector instruments have higher resolution and greater mass range than quadrupole instruments, but they require larger vacuum pumps and often scan more slowly. The typical mass range is to m/z 5000, but this may be extended to m/z 30,000. M agnetic sector instruments are often used in

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series with an electric sector, for high resolution and tandem mass spectrometry experiments.

Electric Sector/Double Focusing Mass Spectrometers. An electric sector consists oftwo concentric curved plates. A voltage is applied across these plates to bend the ion beam as it travels through the analyzer. The voltage is set so that the beam follows the curve of theanalyzer. The radius of the ion trajectory (r) depends upon the kinetic energy of the ion (V) and the potential field (E) applied across the plates.

r=2 VE

Time-of-Flight. The time-of-flight (TOF) mass analyzer separates ions in time as they travel down a flight tube. This is a very simple mass spectrometer that uses fixed voltages and does not require a magnetic field. The greatest drawback is that TOF instruments have poor mass resolution, usually less than 500. These instruments have high transmission efficiency, no upper m/z limit, very low detection limits, and fast scan rates.

Quadrupole Ion Trap. The Quadrupole ion storage trap mass spectrometer (QUISTOR) is a recently developed mass analyzer with some special capabilities. Several commercial instruments are available and this analyzer is becoming more popular. QUISTORs are very sensitive, relatively inexpensive, and scan fast enough for GC/MS experiments. The sensitivity of the QUISTOR results from trapping and then analyzing all the ions produced in the source. Since all the ions are detected, the S/N is high.

Ion Cyclotron Resonance. The Ion Cyclotron Resonance (ICR) mass spectrometer uses a superconducting magnet to trap ions in a small sample cell. This type of mass analyzer has extremely high mass resolution (ca. 109) and is also useful for tandem mass spectrometry experiments. These instruments are very expensive and are typically usedfor specialized research applications. The ICR traps ions in a magnetic field that causes ions travel in a circular path . This is similar to the path of an ion in a magnetic sector, but theions are not traveling as fast and the magnetic field is stronger. As a result the ions are contained in the small volume of the trap.

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The ion’s cyclotron frequency ω, is the angular frequency* of an ion's orbit. This frequency is determined by the magnetic field strength (B) and the m/z value of the ion.

ω=Bem/z

DetectorsA device that detects the ions produced in the mass spectrometer and produces a measurable signal, generally an electronic signal. In most detectors, this signal is amplified. Common types include the Faraday cup, the electron multiplier, the microchannel plate detector and the Daly photomultiplier detector. Faraday Cup Photomultiplier Conversion Dynode Array Detector Charge (or Inductive) Detector Electron Multiplier

Faraday CupA Faraday cup involves an ion striking the dynode (BeO, GaP, or CsSb) surface which causes secondary electrons to be ejected. This temporary electron emission induces a positive charge on the detector and therefore a current of electrons flowing toward the detector. This detector is not particularly sensitive, offering limited amplification of signal, yet it is tolerant of relatively high pressure.

Photomultiplier Conversion DynodeThe photomultiplier conversion dynode detector is not as commonly used at the electron multiplier yet it is similar in design where the secondary electrons strike a phosphorus screen instead of a dynode. The phosphorus screen releases photons which are detected by the photomultiplier. Photomultipliers alsooperate like the electron multiplier where the striking of the photon onscintillating surface results in the release of electrons that are then amplified using the cascading principle. One advantage of the conversion

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dynode is that the photomultiplier tube is sealed in a vacuum, unexposedto the environment of the massspectrometer and thus the possibility of contamination is removed. This improves the lifetimes of these detectors over electron multipliers. A five-year or greater lifetime is typical, and they have a similar sensitivity to the electron multiplier.

Array DetectorAn array detector is a group of individual detectors aligned in an arrayformat. The array detector, which spatially detects ions according to their different m/z, has been typically used on magnetic sector mass analyzers. Spatially differentiated ions can be detected simultaneously by an array detector. The primary advantage of this approach is that, over a small mass range, scanning is not necessary and therefore sensitivity is improved.

Charge (or Inductive) DetectorCharge detectors simply recognize a moving charged particle (an ion) through the induction of a current on the plate as the ion moves past. Atypical signal is shown in Figure. This type of detection is widely usedin FTMS to generate an image current of an ion. Detection is independentof ion size and therefore has been used on particles such as whole viruses.

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Electron MultiplierPerhaps the most common means of detecting ions involves an electron multiplier which is made up of a series (12 to 24) of aluminum oxide (Al2O3 ) dynodes maintained at ever increasing potentials. Ions strike the first dynode surface causing an emission of electrons.These electrons are then attracted to the next dynode held at a higher potential and therefore more secondary electrons are generated. Ultimately, as numerous dynodes are involved, a cascade of electrons is formed that results in an overall current gain on the order of one million or higher.

Vacuum in the Mass SpectrometerAll mass spectrometers need a vacuum to allow ions to reach the detectorwithout colliding with other gaseous molecules or atoms. If such collisions did occur, the instrument would suffer from reduced resolution and sensitivity. Higher pressures may also cause high voltages to discharge to ground which can damage the instrument, its electronics, and/or the computer system running the mass spectrometer. An extreme leak, basically an implosion, can seriously damage a mass spectrometer by destroying electrostatic lenses, coating the optics withpump oil, and damaging the detector. In general, maintaining

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a good vacuum is crucial to obtaining high quality spectra.

Data System:The final component of a mass spectrometer is the data system. This partof the instrument has undergone revolutionary changes in the past twentyyears. It has evolved from photographic plates and strip chart recordersto data systems that control the instrument, acquire hundreds of spectrain a minute and search tens of thousands of reference spectra to identify an unknown.

Interpretation:Although mass spectrometry is a very sensitive instrumental technique, there are other techniques with picogram detection limits. In addition to sensitivity, however, mass spectrometry also is also useful for identifying the chemical structure of this picogram sample.Since the mass spectrum is a fingerprint of the molecular structure, comparison to a computer databases can be used to identify an unknown compound. This is often done using Probability Based Matching (PBM), a popular pattern recognition technique.

INTERPRETATION OF MASS SPECTRUMMass spectrumIt is a simple graph between the abundance of an ion along Y-axis against its mass-to-charge ratio along X-axis. A mass spectrum contains a large number of peaks some are small and some are large, these are Molecular ion peak.

The peak of an ion formed from the original molecule by electron ionization, by the loss of an electron, or by addition or removal of an anion or cation and also known as parent peak, radical peak.

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Fragmentation peaks:- The peaks observed by fragments of compounds.

Base peak:- The most intense ion in a mass spectrum. The abundance of this ion is used as the base fromwhich to normalise the relative abundances of the remaining peaks in thespectrum and is given a nominal value of 100%.

Isotopic peaksPeaks observed due to isotopes like in case of carbon with M+. M+1 peak also observed.

Rules for interpretation of mass spectrumFollowing are the main rules for interpretation

Molecular ion peakIf present it will be highest peak in all isotopic peaks.

DBR CalculationsDouble bond or ring calculations tell us about how many rings or double bonds are present ina compound.DBR= C-H/2+N/2+1C= number of carbon atomsH= number of hydrogen atomsN= number of nitogen atoms

Nitrogen RuleThe nominal molecular weight of a compound will have an even-number value if there are nonitrogen atoms, or an even number of nitrogen atoms, present in the molecule. This holds forcompounds containing C, H, O, P, S, Si, or halogen atoms. Even-electron fragment ionscontaining an even number of nitrogen atoms occur at odd-number m/z values. Conversely, ifthere are an odd number of nitrogen atoms, the nominal molecular weight will be an oddnumber and even-electron ions containing an odd number of nitrogen atomsoccur at even number m/z values.

Isotopic effectMass spectrum can easily be drawn but there are some factors which make the spectra

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complicated, one of these is isotopic effect.

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CHAPTER 3

EXPERIMENTAL WORK

1) COLOUR AND APPEARANCE :- The colour testing was done using Lovibond Tintometer Model F.

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Operating process:-The Lovibond Tintometer is a visual colorimeter that allows matching of samples against Lovibond Colour standards - a series of accurately calibrated coloured glasses in each of the colours red, yellow and blue, going from very pale to dark. It is arranged with two adjacent fields of view, seen through the viewing tube, so that the product in the sample field and a white reflective surface in the comparison field are observed side by side, suitably illuminated. The Lovibond colour standards are introduced intothe comparison field by a simple system of sliding racks, allowing the user to compare the colour of the sample with the standards. A series ofneutral glasses in racks is also supplied; these can be introduced into the sample field to dull the colour of products which are too bright to obtain a good colour match using Lovibond Red,Yellow or Blue glasses. The racks are adjusted until a visual colour match is found for the sample and its colour can then be expressed in Lovibond units.

Mentha oil Colourless , pale yellowor greenish yellow

Palmarosa oil Light yellow to yellow

2) CONGEALING POINT (APPLICABLE TO MENTHA OIL ONLY):-On the other hand, the determination of the congealing point is usually applied in cases where the essential oil consists mainly of one molecule, such as the oil of cloves that contains about 90% of eugenol. Such a test enables the evaluation of the percentage amount of the abundant compound. At congealing point, crystallization occurs accompanied by heat liberation, leading to a rapid increase in temperature which is then stabilized at the so-called congealing point.

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The congealing point of a liquid is the temperature at which it solidifies upon cooling.Congealing point ( mentha oil) = (21-23)0C

3) REFRACTIVE INDEX TEST:- The test was performed on a standard Abbe Refractometer. The standard equipment of the Abbe refractometer includes the standard prism body which is suitable for individual measurement of liquid, solid, and plastic substances. 0.05 ml of a liquid sample are sufficient for one measuring operation. This liquid is situated as a thin film between the measuring and lighting prisms.

A mirror on the prism body illuminates the field of vision with daylightor with light from afilament lamp.The flow prism body for continuously flowing, also for easily volatile liquids is attached like the standard prism body to the instrument. It consists of the measuring and lighting prisms. Both are tightly screwed with each other while a plastic foil has been laid between.

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This results in a small space above the measuring face through which theliquids are flowing. The temperature of the prism body can be controlled.Its separate light source 6 V 1.8 W can easily be attached with a lockable base in front of the measuring prism so that measurement with transmitted and with reflected light is possible.

Sample oil Refractive indexMentha oil 1.4578Palmarosa oil 1.4701

4) SPECIFIC GRAVITY TEST OR RELATIVE DENSITY:- This test was performed using electronic balance, a minute bottle for sample, petroleum etherfor washing bottle.Weight of bottle = 15.5709Weight of mentha oil =4.4327Weight of palmarosa oil =4.361Weight of water (reference) =4.9388

SG= WEIGHTOFOILSAMPLEWEIGHTOFWATER

OIL SAMPLE SPECIFIC GRAVITY(270 C)Mentha oil 0.8975

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Palmarosa oil 0.833

5) OPTICAL ROTATION BY POLARIMETRY: The optical rotation is the angle by which the plane of linear polarizedlight is rotated when passing through the sample. Depending on the direction in which the plane of light is rotated thesubstances are called dextrorotatory (clockwise) or levorotatory(counterclockwise). The view of the observer is towards the lightsource. Dextrorotation is designated (+) and levorotation is designated (-).

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Oil sample Optical rotationMentha oil -350

Palmarosa oil +0.80

6) SOLUBILITY OF ESSENTIAL OILS:-Solubility of mentha oil :-soluble in 1:4 to 6 volume in 70% alcoholSolubility of palmarosa oil :- soluble in 1.2 to 3.5 volume in 70% alcohol

7) GLC Of Mentha Oil:-Name of constituent Quantity (in %)limonene 1.56Menthol 76.781Menthone 7.463-octanol 1.61α-Pinene 0.47β-Pinene 0.56Menthyl acetate 1.12Iso-menthone 3.56Neo-menthol 1.93β-myrcene 0.4isopulegol 1.01

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isomenthol 0.15piperitone 0.46Terpine-4-ol 0.67

8) GLC Of Palmarosa OilName of constituent Quantity (%)Geraniol 79.249Geranyl acetate 9.047linalool 3.807myrcene 0.1farnesol 2.01Geranyl hexanoate 0.8ocimene 1.8Geranyl butyrate 0.2nerol 0.2Beta-caryophyllene 2.6

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CHAPTER 4

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RESULTS AND DISCUSSIONS

1) The sample of palmarose oil was found to be good, unadulterated,having light yellow colour and smelling like rosaceous with a characteristicgrassy background.

2) The sample of mint was unadulterated having colorless/pale yellow colour having characteristic strong minty, followed by cooling sensation.3) The observations were taken under standard temperature and pressure.

4) We find that menthol(76.7) was the major component of mentha oil having the following properties.

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Menthol is an organic compound made synthetically or obtained from cornmint, peppermint or other mint oils. It is a waxy, crystalline substance,clear or white in color, which is solid at room temperature and melts slightly above. 

Formula: C10 H20 O IUPAC ID: (1R,2S,5R)-2-isopropyl-5-methylcyclohexanol Melting point: 31 °C Molar mass: 156.27 g/mol Boiling point: 212 °C Density: 890.00 kg/m³

Menthone is a naturally occurring organic compound with a molecular formula C₁₀H₁₈O.

l-Menthone , is the most abundant in nature of the four possiblestereoisomers. Menthone is a monoterpene and a ketone.

Boiling point: 207 °C Molar mass: 154.25 g/mol Formula: C10 H18 O Density: 895.00 kg/m³

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Linalool is a naturally occurring terpene alcohol chemical found inmany flowers and spice plants with many commercial applications,the majority of which based on its pleasant scent. 

Formula: C10H18O Molar mass: 154.25 g/mol Density: 858.00 kg/m³

Limonene is a colourless liquid hydrocarbon classified as a cyclic terpene. The more common D-isomer possesses a strong smell of oranges. 

Formula: C10H16 Boiling point: 176 °C Density: 841.10 kg/m³ IUPAC ID: 1-methyl-4-(1-methylethenyl)-cyclohexene Molar mass: 136.24 g/mol Melting point: -74.35 °C

Pinene (C10H16) is a bicyclic monoterpene chemical compound. There are two structural isomers ofpinene found in nature: α-pinene and β-pinene. As the name suggests, both forms areimportant constituents of pine resin; they are also found in the resins of many other conifers, as well as in non-coniferous plants.Table2

Properties

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Molecular formula C10H16

Molar mass 136.24 g/mol

Appearance Liquid

Density 0,86 g·cm−3 (alpha, 15 °C)

Melting point −62–−55 °C (alpha)

Boiling point 155–156 °C (alpha)

Solubility in water

Practically insoluble in water

The main constituent of palmarosa oil is geraniol (79.249) having properties. Geraniol is a monoterpenoid and an alcohol. It is the primary part of rose oil, palmarosa oil, and citronella oil.

Table 3

Properties

Molecular formula C10H18O

Molar mass 154.25 g mol−1

Density 0.889 g/cm3

Melting point −15 °C (5 °F; 258 K)

Boiling point 230 °C (446 °F; 503 K)

~ 62 ~

Solubility in water 686 mg/L (20 °C)

Geranyl acetate is a natural organic compound that is classified as a monoterpene. It is a colorless liquid with a pleasant floral or fruity rose aroma. Its condensed liquid has a sightly yellow color.

Molar mass: 196.29 g/molDensity: 916.00 kg/m³Boiling point: 245 °C

Nerol is a monoterpene found in many essential oils suchas lemongrass and hops. It was originally isolated from neroli oil, hence its name. This colourless liquid is used in perfumery. 

Formula: C10H18OMolar mass: 154.25 g/molDensity: 881.00 kg/m³

Caryophyllene  or (−)-β-caryophyllene, is a natural bicyclic sesquiterpene that is a constituent of many essential oils, especially clove oil.

Properties

~ 63 ~

Molecular formula

C15H24

Molar mass 204.36 g/mol

Density 0.9052 g/cm3

Boiling point 262-264 °F; 129-130 °C (14 mm Hg)

CHAPTER 5

CONCLUSIONS

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1) The samples were found to be in good condition, unadulterated.

2) Adulteration usually is accomplished by adding a similar but cheaper oil, such as cornmint oil (Mentha arvensis) , or by diluting the oil with various solvent oils.

3) Despite then value of identifying and quantifying major components like menthol, methone and methyl acetate, dependable identification and quantification is difficult because each of these is represented by several stereoisomers. Menthol, for example, has three chiral centers, for a total of eight stereoisomers, making chromatographic separation difficult.

4) India is the largest exporting country for mentha oil. Mint products including mint oils, menthol crystals and menthol powder, is the single largest product group in the export basket accounting for 22-27% of spices export from India.

5) The palmarosa essential oil contains valuable active pharmaceutical ingredients such as geranylgeraniol, limonene, dihydrocitronellol and sesquiterpenes. The geranylgeraniol and limonene are valuable

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aroma chemicals widely used in the fragrance industries and the oil exhibited high level inhibitory potency against Saccharomyces sp,Candida albicans and Candida tropicalis.

6) The grass yield more oil recovery if dried for nearly one week. The steam distillation seems to be better than the other type of extraction. Palmarosa yields 15 to 20 tonnes of herbage per ha in a year with an oil yield of 50-60 Kg per year.

7) Oil ofpalmarosa, an important perfimery oil, is derived born the plant Cymbopogonmarfinii, stapt fm. Gramineae and exists in two distinct varieties, namely, motiu and sofia. Oil of palmarosa (ex var. motia), is much sought after in perfumery, whereas the comparatively cheaper oil of gingergrass (ex var. sofia) is not. It is, therefore, likely that the latter is used to adulterate the former.

REFERENCES~ 66 ~

Bohra, P., Vaze, A.S., Pangarkar, V.G., Taskar, A., 1994. Adsorptive recovery of water soluble essential oil components. J. Chem. Technol. Biotechnol. 66, 97–102.

Clevenger, J.F., 1928. Apparatus for the determination of volatile oil. J. Am. Pharm. Assoc. 17, 345–349.

Davies, N.W., 1990. Gas chromatographic retention indices of monoterpenes and sesquiterpenes on methyl silicone and carbowax 20Mphases. J. Chromatogr. 503, 1–24.

Fleisher, A., 1990. The poroplast extraction technique in the flavor and fragrance industry. Perfum. Flavor. 15, 27–34.

Fleisher, A., 1991. Water-soluble fractions of the essential oils. Perfum. Flavor. 16, 37–41.

Machale, K.W., Niranjan, K., Pangarkar, V.G., 1997. Recovery of dissolved essential oils from condensate waters of basil and Mentha arvensis distillation. J. Chem. Technol. Biotechnol. 69,362–366.

Bahl, J.R., Bansal, R.P., Garg, S.N., Naqvi, A.A., Luthra, R., Kukreja, A.K. and Kumar, S. 2000. Qualitative evaluation of the essential oils of the prevalent cultivars of commercial mint species Mentha arvensis, M. spicata, M. pipermint, M. cardiaca, M. citrata and M. viridis cutivated in indo-gangetic plains. Journal of Medicinal and Aromatic Plants Sciences 22: 787-797.

Lawrence, B.M.1994. Progress in essential oils. Perfumer & Flavourist 19:58-60.

E. Stahl, Thin layer chromatography, 2nd edition, Springer-Verlag Berlin, Reprint 1988

Davis, R. Mass Spectrometry/Analytical Chemistry by Open Learning; Wiley: New York,

1987.

Harrison, W.W.; Hess, K.R.; Marcus, R.K.; King, F.L. Anal. Chem. 1986,58, 341A-356A.

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